Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system

The histone H3K4 demethylase JARID1A directly interacts with haematopoietic transcription factor GATA1 in erythroid cells through its second PHD domain

Chromatin remodelling and transcription factors play important roles in lineage commitment and development through control of gene expression. Activation of selected lineage-specific genes and repression of alternative lineage-affiliated genes results in tightly regulated cell differentiation transcriptional programmes. However, the complex functional and physical interplay between transcription factors and chromatin modifying enzymes remains elusive. Recent evidence has implicated histone demethylases in normal haematopoietic differentiation as well as in malignant haematopoiesis. Here we report an interaction between H3K4 demethylase JARID1A and the haematopoietic-specific master transcription proteins SCL and GATA1 in red blood cells. Specifically, we observe a direct physical contact between GATA1 and the second PHD domain of JARID1A. This interaction has potential implications for normal and malignant haematopoiesis

Mechanisms controlling anaemia in Trypanosoma congolense infected mice.

Trypanosoma congolense are extracellular protozoan parasites of the blood stream of artiodactyls and are one of the main constraints on cattle production in Africa. In cattle, anaemia is the key feature of disease and persists after parasitaemia has declined to low or undetectable levels, but treatment to clear the parasites usually resolves the anaemia. The progress of anaemia after Trypanosoma congolense infection was followed in three mouse strains. Anaemia developed rapidly in all three strains until the peak of the first wave of parasitaemia. This was followed by a second phase, characterized by slower progress to severe anaemia in C57BL/6, by slow recovery in surviving A/J and a rapid recovery in BALB/c. There was no association between parasitaemia and severity of anaemia. Furthermore, functional T lymphocytes are not required for the induction of anaemia, since suppression of T cell activity with Cyclosporin A had neither an effect on the course of infection nor on anaemia. Expression of genes involved in erythropoiesis and iron metabolism was followed in spleen, liver and kidney tissues in the three strains of mice using microarrays. There was no evidence for a response to erythropoietin, consistent with anaemia of chronic disease, which is erythropoietin insensitive. However, the expression of transcription factors and genes involved in erythropoiesis and haemolysis did correlate with the expression of the inflammatory cytokines Il6 and Ifng. The innate immune response appears to be the major contributor to the inflammation associated with anaemia since suppression of T cells with CsA had no observable effect. Several transcription factors regulating haematopoiesis, Tal1, Gata1, Zfpm1 and Klf1 were expressed at consistently lower levels in C57BL/6 mice suggesting that these mice have a lower haematopoietic capacity and therefore less ability to recover from haemolysis induced anaemia after infection

Molecular mechanisms in haematological malignancies

Haematopoiesis requires the constant production of large numbers of peripheral blood cells. This process is under tight control of transcription factor networks as well as cytokines, growth factors and hormones. We will review the importance of transcription factors in programming the haematopoietic lineage commitment and the role of the microenvironment and the corresponding cellular sensitivity to ensure production of mature functional cells in response to the physiological demand. Understanding the molecular mechanism of this complex process gives the opportunity to identify the underlying molecular deregulation in haematopoietic malignancies. The different levels of deregulation include hyperproliferation, block in differentiation and sensitivity to growth factors. In this review, leukaemic transformation is selected to give evidence of cell signalling deregulation. The clinical implications will be reviewed in the context of the potential opportunities in the future to identify specific therapeutic patient groups that can be defined using prognostic and predictive biomarkers.peer-reviewe

Development of flow focusing device for the visualization of leukocyte rolling adhesion

La microfluídica es un área de la microtecnología basada en chips de PDMS que está siendo utilizada cada vez más en multitud de aplicaciones. Una de estas aplicaciones es la investigación biomédica. La microfluídica o “Lab on a Chip” se ha convertido en una manera de realizar experimentos biomédicos y diagnósticos de una manera barata, rápida y eficaz. Cuando se realizan estudios sobre la extravasación leucocitaria utilizando chips microfluídicos, podemos observar la inconsistencia en la trayectoria de rodadura de los leucocitos debido a un flujo laminar. En este trabajo de fin de grado presentamos un método para centrar la interfaz de células en el centro de canal microfluídico. Cuando las células circulan por los sistemas microfluídicos, las células tienden a circular de manera aleatoria por los canales. Por tanto, con el sistema propuesto en este trabajo, dichas células serán redirigidas a la porción central del canal con el fin de recrear el fenómeno de rodadura presente en nuestro sistema circulatorio y así obtener información más detallada. Los resultados de este trabajo muestran la utilidad y la versatilidad de este dispositivo para experimentos relacionados

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the haematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable. In contrast, conditional deletion of Syk in other haematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that is dependent on CLEC-2 and Syk. These studies demonstrate that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behaviour in vitro

Ribosomal proteins in zebrafish haematopoiesis and human disease

PhDSeveral congenital disorders of human haematopoiesis including Diamond- Blackfan anaemia result from heterozygous loss of genes involved in ribosome biogenesis. Further, hemizygosity for ribosomal protein gene RPS14 has been implicated in the pathogenesis of myelodysplastic syndrome with loss of 5q, suggesting that genes involved in ribosome biogenesis may act as both haploinsufficient tumour suppressors and regulators of normal haematopoiesis. Ribosome biogenesis is highly conserved through evolution and readily studied in simple organisms such as yeasts. However the zebrafish provides a wellestablished genetic model system which is ideally suited to rapid assessment of vertebrate haematopoiesis. I have therefore used the zebrafish to study genes involved in ribosome biogenesis and their effects on developmental haematopoiesis relevant to human disease. Presented in this work is investigation of the effect of disruption of 4 genes known to be involved in ribosome biogenesis on zebrafish haematopoiesis. Firstly, I describe a gene, Dead-box 18 (ddx18), identified in a forward genetic screen, whose disruption results in defective haematopoiesis and embryonic lethality. Secondly, I have studied the effects of loss of zebrafish orthologues of the human nucleophosmin gene (NPM1), the most frequently mutated gene in human acute myeloid leukaemia. Loss of Npm1 resulted in aberrant numbers of myeloid cells. Heterologous overexpression of mutated NPM1(NPMc+) resulted in increased production of haematopoietic stem cells suggesting a role for NPMc+ in pathogenesis of AML. Finally, I have shown that loss of Rps14 and Rps19 result in anaemia in developing zebrafish and have investigated p53-independent mechanisms for this effect. The findings described herein demonstrate that disruption of normal ribosome biogenesis frequently results in abnormal developmental haematopoiesis. Further genetic assessment of these tissue-specific pathways deregulated by loss of normal ribosome function may represent an important common mechanism underlying the pathogenesis of congenital and acquired disorders of haematopoiesis, and may provide novel pathways for therapeutic targeting

HoxA3 is an apical regulator of haemogenic endothelium.

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells

Preliminary investigation of the effects of long-term dietary intake of genistein and daidzein on hepatic histopathology and biochemistry in domestic cats (Felis catus)

Dietary isoflavones have been hypothesised to play a role in hepatic veno-occlusive disease in captive exotic felids, although empirical evidence is lacking. This study aimed to investigate the effect of long-term (>1 year) dietary genistein and daidzein exposure on the hepatic biochemistry and histology of domestic cats. Individual cats were assessed for hepatic enzyme and bile acid production before and after the removal of isoflavones from their diet in the treatment group (n=4), and at the same times in unexposed control animals (n=7). No significant differences were detectable in hepatic biochemistry between treatment and control groups, and all serum values were within the normal reference ranges for domestic cats. Additionally, treatment animals demonstrated slightly greater areas of fibrosis surrounding hepatic venules than control animals, but this difference was not statistically significant. On the basis of the results presented, dietary isoflavones, at the current dose and duration of exposure do not appear to modulate hepatic enzyme production or histological parameters