Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

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.

HIF1α-induced PDGFRß signaling promotes developmental HSC production via IL-6 activation.

Hematopoietic stem cells (HSCs) have the ability to both self-renew and differentiate each of the mature blood cell lineages and thereby reconstitute the entire blood system. Therefore, HSCs are therapeutically valuable for treatment of hematological malignances and bone marrow failure. We showed recently that transient glucose elevation elicited dose-dependent effects on HSCs through elevated metabolic activity and subsequent reactive oxygen species-mediated induction of Hypoxia-Inducible Factor 1α (Hif1α). Platelet-Derived Growth Factor B (pdgfb), a Hif1α-target, and its receptor, pdgfrb, were significantly upregulated in response to metabolic stimulation. Although the function of PDGF signaling is well established in vascular development, its role in hematopoiesis is less understood. Exposure to either a pan-PDGF inhibitor or a PDGFRß-selective antagonist in the context of Hif1α stimulation blocked elevations in hematopoietic stem and progenitor cell (HSPC) formation as determined by runx1;cmyb whole-mount in situ hybridization (WISH) and HSPC-reporter flow cytometry analysis. Similar results were observed for morpholino (MO) knockdown of pdgfrb or dominant-negative pdgfrb expression, indicating that PDGFRß signaling is a key downstream mediator of Hif1α-mediated induction of HSPCs. Notably, overexpression of Pdgfb ligand enhanced HSPC numbers in the aorta-gonado-mesonephros (AGM) at 36 hours postfertilization (hpf) and in the caudal hematopoietic tissue at 48 hpf. A survey of known PDGF-B/PDGFRß regulatory targets by expression analysis revealed a significant increase in inflammatory intermediates, including Interleukin 6 (IL-6) and its receptor (IL-6R). MO-mediated knockdown of il6 or chemical inhibition of IL-6R antagonized the effect of Pdgfb overexpression. Furthermore, epistatic analysis of IL-6/IL-6R function confirmed activity downstream of Hif1α. Together, these findings define a Hif1α-regulated signaling axis mediated through PBFGB/PDGFRß and IL-6/IL-6R that acts to control embryonic HSPC production.

Developmental Vitamin D Availability Impacts Hematopoietic Stem Cell Production.

Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8(IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.

The Central Nervous System Regulates Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling.

Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers.

Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease.

Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish.

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.

Cannabinoid receptor signaling regulates liver development and metabolism.

Endocannabinoid (EC) signaling mediates psychotropic effects and regulates appetite. By contrast, potential roles in organ development and embryonic energy consumption remain unknown. Here, we demonstrate that genetic or chemical inhibition of cannabinoid receptor (Cnr) activity disrupts liver development and metabolic function in zebrafish (Danio rerio), impacting hepatic differentiation, but not endodermal specification: loss of cannabinoid receptor 1 (cnr1) and cnr2 activity leads to smaller livers with fewer hepatocytes, reduced liver-specific gene expression and proliferation. Functional assays reveal abnormal biliary anatomy and lipid handling. Adult cnr2 mutants are susceptible to hepatic steatosis. Metabolomic analysis reveals reduced methionine content in Cnr mutants. Methionine supplementation rescues developmental and metabolic defects in Cnr mutant livers, suggesting a causal relationship between EC signaling, methionine deficiency and impaired liver development. The effect of Cnr on methionine metabolism is regulated by sterol regulatory element-binding transcription factors (Srebfs), as their overexpression rescues Cnr mutant liver phenotypes in a methionine-dependent manner. Our work describes a novel developmental role for EC signaling, whereby Cnr-mediated regulation of Srebfs and methionine metabolism impacts liver development and function.

Accumulation of the Vitamin D Precursor Cholecalciferol Antagonizes Hedgehog Signaling to Impair Hemogenic Endothelium Formation.

Hematopoietic stem and progenitor cells (HSPCs) are born from hemogenic endothelium in the dorsal aorta. Specification of this hematopoietic niche is regulated by a signaling axis using Hedgehog (Hh) and Notch, which culminates in expression of Runx1 in the ventral wall of the artery. Here, we demonstrate that the vitamin D precursor cholecalciferol (D3) modulates HSPC production by impairing hemogenic vascular niche formation. Accumulation of D3 through exogenous treatment or inhibition of Cyp2r1, the enzyme required for D3 25-hydroxylation, results in Hh pathway antagonism marked by loss of Gli-reporter activation, defects in vascular niche identity, and reduced HSPCs. Mechanistic studies indicated the effect was specific to D3, and not active 1,25-dihydroxy vitamin D3, acting on the extracellular sterol-binding domain of Smoothened. These findings highlight a direct impact of inefficient vitamin D synthesis on cell fate commitment and maturation in Hh-regulated tissues, which may have implications beyond hemogenic endothelium specification.

Cannabinoid Receptor-2 Regulates Embryonic Hematopoietic Stem Cell Development via Prostaglandin E2 and P-Selectin Activity.

Cannabinoids (CB) modulate adult hematopoietic stem and progenitor cell (HSPCs) function, however, impact on the production, expansion, or migration of embryonic HSCs is currently uncharacterized. Here, using chemical and genetic approaches targeting CB-signaling in zebrafish, we show that CB receptor (CNR) 2, but not CNR1, regulates embryonic HSC development. During HSC specification in the aorta-gonad-mesonephros (AGM) region, CNR2 stimulation by AM1241 increased runx1;cmyb(+) HSPCs, through heightened proliferation, whereas CNR2 antagonism decreased HSPC number; FACS analysis and absolute HSC counts confirmed and quantified these effects. Epistatic investigations showed AM1241 significantly upregulated PGE2 synthesis in a Ptgs2-dependent manner to increase AGM HSCs. During the phases of HSC production and colonization of secondary niches, AM1241 accelerated migration to the caudal hematopoietic tissue (CHT), the site of embryonic HSC expansion, and the thymus; however these effects occurred independently of PGE2. Using a candidate approach for HSC migration and retention factors, P-selectin was identified as the functional target of CNR2 regulation. Epistatic analyses confirmed migration of HSCs into the CHT and thymus was dependent on CNR2-regulated P-selectin activity. Together, these data suggest CNR2-signaling optimizes the production, expansion, and migration of embryonic HSCs by modulating multiple downstream signaling pathways.

Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production.

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

Estrogen defines the dorsal-ventral limit of VEGF regulation to specify the location of the hemogenic endothelial niche.

Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17ß-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.

Glucose metabolism impacts the spatiotemporal onset and magnitude of HSC induction in vivo.

Many pathways regulating blood formation have been elucidated, yet how each coordinates with embryonic biophysiology to modulate the spatiotemporal production of hematopoietic stem cells (HSCs) is currently unresolved. Here, we report that glucose metabolism impacts the onset and magnitude of HSC induction in vivo. In zebrafish, transient elevations in physiological glucose levels elicited dose-dependent effects on HSC development, including enhanced runx1 expression and hematopoietic cluster formation in the aorta-gonad-mesonephros region; embryonic-to-adult transplantation studies confirmed glucose increased functional HSCs. Glucose uptake was required to mediate the enhancement in HSC development; likewise, metabolic inhibitors diminished nascent HSC production and reversed glucose-mediated effects on HSCs. Increased glucose metabolism preferentially impacted hematopoietic and vascular targets, as determined by gene expression analysis, through mitochondrial-derived reactive oxygen species (ROS)-mediated stimulation of hypoxia-inducible factor 1α (hif1α). Epistasis assays demonstrated that hif1α regulates HSC formation in vivo and mediates the dose-dependent effects of glucose metabolism on the timing and magnitude of HSC production. We propose that this fundamental metabolic-sensing mechanism enables the embryo to respond to changes in environmental energy input and adjust hematopoietic output to maintain embryonic growth and ensure viability.

Sarcoma embrionario hepático: reporte de un caso / Embryonal sarcoma of the liver: case report

El sarcoma embrionario hepático ocupa el tercer lugar en frecuencia de aparición entre los tumores hepáticos primarios de la edad pediátrica. Su presentación clínica es inespecífica y su apariencia en imágenes es una lesión sólida con componente quístico. Aunque es necesaria la confirmación histológica, el manejo inicial se orienta con base en los hallazgos de las imágenes. En este artículo se presenta el caso de una niña de 9 años de edad con este diagnóstico y se describen las principales características clínicas, histológicas y de imagen.

Embryonal hepatic sarcoma occupies the third place in frequency among hepatic tumors in the pediatric population. Its clinical symptoms are usually non specific, and its appearance in diagnostic images is of a solid mass with a cystic component. Even though a histological confirmation is necessary, the initial management is oriented based on the imaging findings. This article presents a case report of a 9 year old girl with the diagnosis of an embryonal hepatic sarcoma; a description is done of the main clinical, histological and imaging characteristics.

Caracterización de la situación de seguridad alimentaria de la población en condición de desplazamiento forzado: una mirada a través de las familias, estudio de caso Bogotá / Nutricional security characterization of some population in forced displacement a look through the families. Case study in Bogota

La seguridad alimentaria y nutricional de la población en condición de desplazamiento forzado se esta vulnerando, situación que se evidencia en inseguridad y periodos de hambre de las familias, programas asistenciales que no responden a una política estatal clara frente al problema, dificultades en el acceso a la educación, trabajo mal remunerado e inestable, acceso a servicios públicos precarios y asentamiento en lugares de alto riesgo. Las voces de los desplazados no se escuchan y lo que ellos proponen no es prioridad para las instituciones, responsables de atender esta problemática. Este trabajo intenta rescatar esas voces que buscan alternativas para la solución a un problema básico que es la subsistencia de las familias desplazadas de Bogotá, el combate a la inseguridad y la lucha cotidiana por sobrevivir al hambre a corto plazo que permanentemente los aqueja

Evaluación del diagnóstico de pancreatitis aguda por los índices de amilasa y creatinina - H.R.S.B, Bogotá 1985-1988 / Diagnosis of acute pancreatitis by amilase levels

Se seleccionaron 11 pacientes que ingresaron al servicio de urgencias de HRSB con diagnóstico inicial de PA, durante el período comprendido entre Julio de 1987. Con la dosificación de amilasa y creatinina en sangre y orina se determinaron los índices de amilasuria/creatinuria, depuración amilasa/creatinina y amilasemia en todos los pacientes.A través de la evolución clínica y los estudios paraclínicos se comprobó el diagnóstico de PA en 6 pacientes,descartándose en los restantes,conformando de ésta manera dos grupos, en los cuales comparamos la sensibilidad y especificidad (80 por ciento) para los 3 índices,en tanto que el índice depuración amilasa/creatinina mostró tener la mayor sensibilidad (66 por ciento).Se concluyó que la amilasemia y el índice amilasuria/creatinuria, no demostraton tener ninguna ventaja sobre el índice depuración amilasa/creatinina,por lo cual sugerimos a este último, como el método paraclínico a utilizar en el diagnóstico de la PA en nuestra institución