2 Material and Methods
3.3 Manuallycounted ﬂuorescentcells
I would like to thank Prof. Bernd Pelster for enabling this masters thesis. My sincere thanks applies to Prof. Thorsten Schwerte for the excellent supervision of this work.
Of course I would like to thank Dr. Renate Kopp, Dr. Nikolaus Medgyesy and Dr. Margit Egg for technical support and the pleasant atmosphere in the laboratory.
Finally my thanks goes to all the members of the Institute of Zoology which helped with words and deeds.
illustration not visible in this excerpt
The transcription factor GATA-1 is essential for the development of the erythroid cell lineage in vertebrates. In this article we introduce a method to easily determine the approximately development status of red blood cells and the progression of blood formation by intensity of ﬂuorescence in GATA1/Ds-Red marked zebraﬁsh. We classiﬁed the blood cells on the basis of their ﬂuorescence intensity in 5 intensity stages (IS) with the brightest in IS 1. The comparison with our erythropoietin (Epo) data showed a noticable correlation between GATA-1, Epo mRNA and EPO protein level. Between 2 and 3 dpf we observed a major increase in blood cell concentration to circa 1200 cells*nl − 1 , until 15 dpf this value decreased to about the half. The appearance of IS 1 cells correspond approximately to the peaks in Epo cRNA copies and the highest values in EPO protein emerged about 1 day later. Our data show that the blood cell concentration, Epo and Gata-1 expression in zebraﬁsh larvae is subjected to large ﬂuctuations in the ﬁrst few days of development.
The zebraﬁsh Dani o rerio, also known as Brachidani o rerio, Cyprinu s reri o and others, is a omnivorous, tropical ﬁsh of the family Cyprinida e and was at ﬁrst described by Hamilton in 1822. Its natural range within Asia are slow moving or stagnant water bodies in India, Bangladesh, Nepal and Pakistan [1, as cited in 2]. In the past few decades the zebraﬁsh became an important model organism for genetical, developmental and physiological studies. Fish are vertebrates and thus the genetic program is more similar to that of mammals than invertebrate models like the fruitﬂy (Drosophil a melanogaster). Because of this relationship most of the zebraﬁsh genes have human orthologs 3. Due to its short generation time of approximately 3 month, the rapid development and the transparency up to adulthood this tropical teleost is predestined for i n viv o hematologic studies and digital imaging techniques. Especially digital imaging is a gentle, non-invasive and thereby seminal method for researcher who are working with transparent animals. The combination with ﬂuorescent reporter genes allows real time imaging of gene expression and cell migration studies over their whole lifespan 4.
1.1 Zebraﬁsh hematopoiesis
Zebraﬁsh hematopoiesis can be classiﬁed into a primitive (embryonic) and deﬁnitive (fetal and adult) part. The primitive hematopoiesis produces primarily erythrocytes and some macrophages, whereas the deﬁnitive part is responsible for cells of the erythroid, lymphoid and myeloid lineage 5. The embryonic hematopoiesis occurs in a region called ”intermediate cell mass” (ICM), which is located dorsally to the yolk tube. The ICM arises from posterior-lateral mesoderm at the 5-somite stage (≈ 11,5 hpf) and contains, amongst others, primitive hematopoietic precursors and gives rise to the endothelia of the major trunk vessels 6, 7.
The ﬁrst markers of deﬁnitive hematopoiesis are detected in the ICM after about 30 h. It has been shown that a few hematopoietic cells exist at other locations like the ventral wall of the dorsal aorta, which may be the ﬁrst site of deﬁnitive hematopoiesis, and the ”ventral vein region”, a region in the tail ventral to the axial vein. This ventral vain region (VVR) comprises of the posterior part of the ICM and is proposed to be an larval site for endothelial tissue development [5, 8. The major site for hematopoiesis through adulthood is the kidney. Additionally, Willett et al. found circulating erythroblasts and immature erythrocytes associated to the heart endocardium from 24 hpf onward, which could be conﬁrmed by other authors. There is evidence to suggest that the heart is a blood forming organ between the disappearance of the ICM and the onset of deﬁnitive hematopoiesis in the pronephros at about hour 96 pf 9, 10.
Zebraﬁsh erythropoiesis consists of two waves of red blood cell production, a primitive and a deﬁnitive one. The primitive erythropoiesis generates erythrocytes (EryP) which express embryonic hemoglobin, erythrocytes of the deﬁnitive wave (EryD) synthesise the adult type of globin 11. After 10 somites (≈ 10 hpf) blood precursors express mature erythroid markers such as embryonic globin . Later, at about the 18-somite stage (≈ 15,5 hpf), primordial blood cells are differentiated out of it 121314. The 20 h ICM already contains proerythroblasts and about 24 hpf the ﬁrst blood cells enter the developing circulatory system. Till about 30 hpf more and more cells will be received and the ICM disappears . The circulating proerythroblasts mature to ﬂattened elliptical erythrocytes during the next 4 days 15.
1.2 GATA-1 and Co-factors
1.2.1 The role of GATA-1
GATA-1/2/3 are involved in hematopoiesis, while GATA-4/5/6 regulate heart, gut and lung development in vertebrates . Transcripts encoding GATA-1 are ﬁrst detected at the two to three somite stage (≈ 10,3 hpf) in two stripes of cells ﬂanking paraxial mesoderm which later will fuse to the ICM 16. No GATA-1 expression could be detected in the posterior ICM at 23 hpf and the cells seems to be less differentiated than those in the anterior ICM . Long et al. even reported GATA-1 expression approximately after 8 hpf (mid-gastrula stage) in the ventral region of the embryo .
Due to the fact that zebraﬁsh are vertebrates, the DNA sequence motive GATA is well conserved from ﬁsh to humans . GATA factors typically share the consensus binding sequence WGATAR (W = A or T; R = A or G) and the GATA motive is present in cis -regulatory elements of many erythroid-expressed genes 17, 18. The expression of this zinc ﬁnger protein is limited to erythroid, eosinophil, megakaryocyte, mast cell lineages and multipotential myeloid progenitors. GATA-1, formerly known as GF1, NF-E1 and Eryf-1, was the ﬁrst characterised factor of the six members including GATA family and is normally the most abundant GATA factor in late erythroid differentiation [16, 19, 20. The protein contains two zinc ﬁngers comprising a zinc atom linked to four cysteines per ﬁnger. The carboxyl terminal is responsible for binding the WGATAR recognition sequence, whereas the amino terminal zinc ﬁnger stabilises this interaction and binds several cofactors. The C-terminal tail is responsible for speciﬁc DNA binding and wraps around into the minor groove of the DNA 21, 22. GATA-1 mutants lacking the N-terminal domain do not show transcriptional activity, but are able to restore differentiation of GATA-1 deﬁcient embryonic stem cells in vitro 23.
Studies on GATA-1 deﬁcient mice showed, that the mutation don’t reduce the number of erythroid progenitors, or affect colony-forming potential. Hematopoietic cells lacking GATA-1 are able to enter erythroid lineage, but cease differentiation mostly at the proerythroblast stage. At the same time GATA-2 expression is increased in erythroid progenitors [17, 19]. This may be due to the fact, that GATA-2 partly overlaps in function with GATA-1 . In contrast, mast cell and megacaryocyte lines are able to complete differentiation in absence of GATA-1. A lack of GATA-2 in mice leads to a insuﬃcient number of erythroid progenitors, megacaryocytes and mast cells [17, 24. In GATA-1 deﬁcient zebraﬁsh embryos hematopoietic cells in the ICM were found to differentiate into myelomonocytes . Thus GATA-1 initiates terminal erythroid differentiation and besides suppresses cell growth by suppressing transcription of responsible genes or interference with proteins like the myeloid transcription factor PU.1 25, 26. GATA-1 is even able to reprogram common lymphoid progenitors (CLPs) and granulocyte-monocyte progenitors (GMPs) to form erythroid colonies through its antagonistic effect on PU.1. It has been shown that in hematopoiesis GATA expression is switched from GATA-2 in early hematopoietic progenitors to GATA-1 during terminal differentiation by suppressing GATA-2 gene expression by GATA-1 [17, 26]. A restoration of GATA-1 in GATA-1 deﬁcient cell lines effect a synchronous cell cycle arrest in the G1 phase and a common differentiation about 12 h after induction 27.
GATA-1 may serve as a direct activator of transcription or as a mediator of promoter-enhancer activity and is moreover a regulator of its own promotor in a positive feedback loop 28, 29. Globin and heme enzyme genes for example are target genes of the GATA-1 protein, their activation results in hemoglobin production . Additionally Briegel et al reported a hyper-phosphorylated GATA-1 species preferentially located in the nucleus of avian erythroid progenitor cells after differentiation induction. Before differentiation induction the vast majority of GATA-1 is restricted to the cytoplasm. This indicates that phosphorylation is an important process for translocation of GATA-1 through the nuclear membrane and moreover enhances the DNA binding aﬃnity [22, 25].
1.2.2 GATA-1 related factors
GATA-1 itself is regulated by some other transcription factors. C-Myb for example, a protein regulating growth and differentiation, was predominantly found in immature hematopoietic cells and leads to GATA-1 expression but disappears during differentiation. C-Myb, just like FOG (Friend Of GATA), directly binds to the N-terminal zinc-ﬁnger of GATA-1 [22, 30]. FOG is a nine zinc-ﬁnger containing cofactor protein which is strongly expressed in hematopoietic progenitors, erythroid and megacaryocyte lineages and at low levels in lymphoid and myeloid cell lines . Transfection of c-Myb in undifferentiated ES (erythroid stem) cells leads to an up-regulation of GATA-1 expression . In contrast Takahashi et al showed that c-Myb provoked an transcriptional inhibition of GATA-1 and vice versa . FOG competes against c-Myb for binding to GATA-1 and accelerates erythroid differentiation by enhancing its activity while c-Myb would inhibit it 30, 31.
Two other GATA-1 stimulating transcriptional cofactors are CBP (cAMP response element-binding protein binding protein)and a close relative to CBP, p300. CBP/p300 shows histone acetyltransferase activity and additionally acetylates GATA-1 at two highly conserved lysine rich motifs near the zinc-ﬁnger domains32. Acetylation of histones leads to a more loose chromatin structure, called euchromatin, and thereby afford the binding of polymerases and several transcription factors. A loss of CBP/p300 function yields to a block in cellular differentiation and speciﬁc gene expression . During differentiation the lineage speciﬁc factor FOG is replacing the multifunctional cofactor CBP . Boyes et al reported that the acetylation effects a conformational change and increases both the DNA-binding aﬃnity and the mobility of the protein like studies on mice showed [32, 33]. Contrary to these ﬁndings Hung et al could not provide evidence that acetylation of GATA-1 effects the ability to bind DNA. These different results might be caused by the small varieties between cGATA-1 (chicken GATA-1) and p300 used by Boyes and mGATA-1 (mouse GATA-1) and CBP used by Hung33.
C-Kit, a receptor tyrosine kinase (RTK) recognising the stem cell factor SCF, is responsible for survival, maturation and proliferation of hematopoi etic stem cells and progenitors by balancing the expression of pro-and antiapoptotic signals [34, 35. It may also activate the Epo/EpoR signalling pathway by tyrosine phosphorylation of EpoR [35, 36 and activates EpoR by direct interaction with the cytoplasmatic domain of EpoR . This interaction at the CFUe stage is crucial for erythroid differentiation 37. C-Kit down-regulation during progenitor differentiation is required for later stages of terminal differentiation induced by GATA-1. It has been shown, that GATA-1 causes cell cycle arrest by blocking the c-Kit signaling cascade which normally leads to activation of c-Myc . The c-Myc protein forms a heterodimeric complex with Max, a ubiquitously expressed helix-loop-helix/leucine zipper protein, and is involved in several cell growth and differentiation processes. Northern Blot analysis showed, that the c-Myc gene is already expressed at the two-cell stage, but at very low concentrations. As recently as 20 hpf and during periods of growth and differentiation c-Myc can be detected at higher abundance in zebraﬁsh cells 38. Studies on mice showed, that an overexpression of c-Myc proto-oncogene can immortalise various hematopoietic lineages and lead to erythroleukemia 39.
1.3 Epo and EpoR
Erythropoietin (Epo) is a small globular glycoprotein hormone which is essential for deﬁnitive but not primitive hematopoiesis . Its expression level is modulated by decreased tissue oxygen tension, i.e. triggered by high altitudes or severe anemia 40, 41. In mammals Epo production occurs in the fetal liver and in the kidney after birth 42. It normally circulates at low concentrations in the blood and binds to its receptor (EpoR), a single transmembrane cytokine receptor, which is speciﬁcally expressed on erythroid cells [40, 41]. The bridging of two ligated EpoR monomers by Epo to a symmetrical dimer leads to the activation of the receptor .
The growth factor Epo is known to stimulate the proliferation and differentiation of erythroid progenitors by inhibition of apoptosis 43. Under normoxic conditions the low Epo level allows only a small amount of erythroid progenitors to survive 44. Epo deprivation results in caspase me diated cleavage of GATA-1 and apoptosis of immature erythrocytes 45. During differentiation the erythropoietin receptor is expressed in the burst forming unit-erythroid (BFUe) and, in the highest density, in the colony forming unit-erythroid (CFUe). EpoR deﬁcient mice embryos showed an lethal decrease in CFUe cells, which indicates that erythroid progenitors aren’t able to proliferate or survive without an functional EpoR pathway . The interaction of EPO with its membrane bound receptor leads to a signal transduction from the cell surface to the nucleus. After activation of EpoR two Janus kinase 2 molecules (JAK2) phosphorylate eight cytoplasmatic tyrosine residues of the receptor which allows other signalling proteins to bind [44, 46. Deactivation of the receptor is mediated by the hematopoietic cell phosphatase (HCP) which catalyses JAK2 dephosphorylation .
At least two other types of Epo receptors could be found in mice and humans. The soluble form EpoR-S is lacking the transmembrane domain and is binding Epo in mice, but not in humans. EpoR-T (truncated) lacks about one fourth of its cytoplasmic region and is predominantly expressed in immature erythroid progenitors. It can transmit mitogenic signals, but fails to prevent apoptosis 47.
Chiba et al showed in their studies that GATA-1 is transactivating the EpoR gene in a stage-speciﬁc manner, they concluded that GATA-1 has to be expressed before EpoR appears on the cell membrane. On the other hand EpoR-mediated signalling signiﬁcantly boosts GATA-1 expression 48.
Studies on murine J2E and erythroleukemia (MEL) cell cultures showed that about 4–6 hours after exposure to Epo the GATA-1 mRNA level increased signiﬁcantly and lasted for about 36 h. When cells enter terminal differentiation and hemoglobin production reaches its peak GATA-1 mRNA and protein decreases. This suggests that GATA-1 is not necessary to complete terminal differentiation of red blood cells . Globin-positive cells generated in the absence of Epo are unable to mature and die prematurely and GATA-1 expression is greatly reduced 49. Imagawa et al found that the GATA sequence at -30bp in the Epo gene is a negative regulatory element for Epo transcription. GATA-1, 2 and 3 bind to the GATA element of Epo gene promotor and negatively regulate gene expression by inhibiting the promotor 50. Binding of GATA-1 to the EpoR promotor in turn leads to an induction of expression .
1.4 Oxygen dependent development
High ﬂexibility is required to survive the variable conditions of temperature and oxygen content especially of tropical aquatic habitats. So ﬁsh developed a variety of morphological, physiological and behavioural adaptations in the course of time. The structure and function of hemoglobin is just one example out of many more. Fish from poorly oxygenated habitats show a higher hemoglobin oxygen aﬃnity caused by some amino acid substitutions compared to ﬁsh from oxygen-rich waters. Other members of the cyprinid genus, e.g. Carassiu s sp., developed an alternative metabolic pathway for anaerobic energy production: fermentation of glucose to ethanol and carbon dioxide 51. Preliminary studies on zebraﬁsh showed that they have a well developed capacity to tolerate low oxygen tensions 52. As adult they can survive 2kPa oxygen tension and tolerate full anoxia for 24 h at embryonic stage 53, 54.
Several studies showed developmental retardation, elevated embryonic lethality and altered gene expression in all oxygen requiring organisms exposed to hypoxic conditions 55. When transferred to hypoxic water (1 mg O2 × l − ) shortly after fertilisation, larvae show a lower heart rate (bradycardia) and end systolic volume than under normoxic conditions but not a lowered red blood cell velocity or end diastolic volume. This may be due to a bigger arterial diameter and the resulting decreased blood ﬂow resistance . Surprisingly Jacob et al reported signiﬁcant tachycardia at 4 dpf when ﬁsh were incubated under hypoxia (PO 2 = 10 k P a) after 20 h of development. Preliminary studies showed that cardiac activity already responds to hypoxia at the time of hatching, although convective oxygen transport becomes crucial not until about two weeks after fertilisation .
The three members including transcription factor family HIF (hypoxiainducible factor) is known to play an essential role in the response to hypoxia. Thereby HIF-1 is the primary factor for hypoxia mediated gene expression,
while the role of HIF-2 is partially unclear and HIF-3 seems to be a suppressor of hypoxia induced gene expression. The HIF-1α subunit protein is continuously produced, but rapidly degraded under normoxic conditions. The mRNA level thereby is not effected by changing oxygen levels. Degradation is mediated by an oxygen-dependent degradation domain (ODD) in which some proline residues are covalently modiﬁed by prolyl hydroxylase enzymes. The von Hippel-Lindau tumour suppressor protein (pVHL) and an E3 ubiquitin ligase bind to the hydroxylated HIF-1 α, the complex built in this way is immediately degraded by proteasomes . In hypoxia, prolyl hydroxylation does not occur and the HIF-1α protein level is stabilised. The protein then is translocated to the nucleus, dimerises with an aryl hydrocarbon receptor nuclear translocator (ARNT), also known as HIF-1β subunit, and binds to the promotor or enhancer region of hypoxia-inducible genes and upregulates their expression . The HIF-1 pathway is established at very early stages of development, HIF-1α mRNA can be already observed at 1 hpf. The most important regulatory sequence of hypoxia inducible genes is located in the so-called hypoxia response element (HRE), a cis -regulatory DNA sequence [44, 59].
The insulin-like growth factor binding protein 1 (IGFBP-1) is another target gene of HIF. IGFBP-1 binds to the insulin-like growth factor, a protein responsible for cell growth, and inhibits its activity. It has been shown that overexpression of IGFBP-1 results in reduced birth-weight of mice and that elevated IGFBP-1 levels can be found in human fetuses suffering from IUGR (intrauterine growth restriction). In zebraﬁsh hypoxia leads to a HIF1 mediated up-regulation of IGFBP-1 and to developmental retardation . In early zebraﬁsh larvae IGFBP-1 is expressed in many tissues but later in development it is restricted to the liver .
1.5 Intention of this study
The primary aim of this study was to apply an easy to accomplish and noninvasive method to determine expression levels of ﬂuorescence marked proteins by means of ﬂuorescence intensity. Therefor GATA-1/DsRed transgenic zebraﬁsh obtained form our own breeding colony were used. Additionally we were interested in the inﬂuence of hypoxia on the GATA-1 and erythropoietin expression in zebraﬁsh embryos and larvae. We hypothesised that the Epo expression and in series GATA-1 expression is up-regulated and the cell count is on a higher level in the hypoxia treated animals.
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- Leopold-Franzens-Universität Innsbruck – Zoologie