Analysis of Ceylon and TC4, two zebrafish mutants with immunodeficiency

Understanding the developmental underpinnings of the immune system is essential to comprehend how defects in this process lead to disease. T cells are a crucial part of the adaptive immune response and perturbation of T cell development leads to illnesses such as leukemia and immunodeficiency. To understand how defects in T cell development result in disease, we use zebrafish because of their many advantageous features. We focus our analysis on two zebrafish mutants lacking T cells that were identified in a screen for mutants with defects in T cells. By identifying the mutant genes and characterizing the mutant phenotypes, we will gain a better understanding of the molecular mechanisms underlying immunodeficiency. Ceylon, a zebrafish mutant that has severely reduced T cells was our first mutant of interest. Our goal is to use mRNA in situ hybridization to observe which hematopoietic cells are affected in the ceylon mutant. In addition to a severe decrease of T cells hematopoietic stem/progenitor cells (HSC/HSPCs) at 6 days post fertilization (dpf) are severely reduced despite the fact that HSPCs at 2 dpf are normal. Based on our observation of normal definitive HSPCs, our hypothesis is that there is a defect specifically in the niche required for HSPC proliferation and/or survival at later ages, causing loss of HSPCs. To identify defects in other tissues that could contribute to defects in the HSPC niche or HSPC development in general, we have also analyzed ceylon for defects in other non-hematopoietic organs and are studying cell proliferation and cell death within the HSC niche to identify the cause of HSC and T cell loss. The second mutant lacking T cells we are studying is known as TC4, complements ceylon and also has a loss of HSPCs. To identify the specific blood cells affected, we are also using double fluorescent in situs on both ceylon and TC4 mutants. In addition, we are employing a positional cloning approach to identify the genetic lesion that causes these defects. The data from our study will lead to important insights into the cause and mechanism of immunodeficiency and other related diseases.

r ANALYSIS OF CEYLON AND TC4, TWOZEBRAFISH MUTANTS WITH IMMUNODEFICIENCY by Daniel Jun-Kit Hu A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree of Bachelor of Science In Biology Approved: ,J�0 Neil 1. Vickers Nikolaus Trede Chair, Department of Biology Supervisor ��,,� l�h� KTOP Darryl L Martha S. Bradley Department Honors Adviser Dean, Honors College May 2009 ABSTRACT of the immune system is essential to Understanding the developmental underpinnings of comprehend defects in this process lead to disease. T cells are a crucial part of comprehend how defects of the adaptive immune response and perturbation perturbation of of T cell development leads to illnesses such leukemia and immunodeficiency. as leukemia immunodeficiency. To understand how defects in T cell development of their many advantageous features. We zebrafish because of result in disease, we use zebrafish zebrafish mutants lacking T cells that were identified identified in a focus our analysis on two zebrafish screen for mutants with defects in T cells. By identifying identifying the mutant genes and characterizing the mutant phenotypes, we will gain a better understanding of of the molecular Ceylon, a zebrafish molecular mechanisms underlying immunodeficiency. immunodeficiency. Ceylon, zebrafish mutant that has severely reduced T cells was our first mutant of of interest. Our goal is to use mRNA in situ situ hybridization hybridization to observe which hematopoietic cells are affected affected in the ceylon ceylon mutant. of T cells hematopoietic stem/progenitor In addition to a severe decrease of stem/progenitor cells (HSC/HSPCs) at 6 days post fertilization fertilization (dpf) are severely reduced despite the fact that HSPCs at 2 dpf of normal definitive definitive HSPCs, our dpf are normal. Based on our observation of hypothesis is that there is a defect defect specifically specifically in the niche required for HSPC proliferation proliferation and/or survival at later ages, causing loss of of HSPCs. To identify identify defects defects in other tissues that could contribute to defects in the HSPC niche or HSPC development development in general, we have also analyzed ceylon ceylon for defects in other non-hematopoietic non-hematopoietic organs and are studying cell proliferation of proliferation and cell death within the HSC niche to identify identify the cause of HSC and T cell loss. The second mutant lacking T cells we are studying is known as TC4, complements ceylon of HSPCs. To identify ceylon and also has a loss of identify the specific specific blood fluorescent in situs cells affected, affected, we are also using double fluorescent situs on both ceylon ceylon and TC4 ii mutants. In addition, posi tional cloning approach approach to identify identify the addition, we are employing a positional genet ic lesion that causes these defects m our study will wi ll lead to important genetic defects.. The data fro from insights into the cause and mechanism mechan ism of immunodefic iency and other related rel ated diseases. immunodeficiency iii ii i TABLE TABLE OF OF CONTENTS CONTENTS A. Problem Problem Under Under Investigation Inves tigation ................................. ...... ........ ...................11 A. BIl .. Methods Methods (Screening) (Scree ni ng) .. . ... .. . .. ......... ... .. ...................... ........ .. ...... ............ .55 B 82. Methods Methods (Wholemount (Wholemount mRNA mRNA In In Situ Situ Hybridization) Hybridization) ........ ............ "................66 B2. Methods (Double (Doub le Fluorescent Fluo rescent mRNA mRNA In In Situ Situ Hybridization) Hybridizatio n) .............................7? B3. Methods Current Results Results .................................................... .......... . '". "_ .... ...........99 C. Current D. Discussion Discussion ................ .. ............•.......... .. . ......... .•......... .•..................... 22 D. E. Future Direction .............. ... '" ..... .. •... ... .... ... ......... .. ........ ....................... 24 Conclusion .. .. ....... ... .... .................................... , .................... , ...... .. .. .25 F. Conclusion G. Bibliography Bibli ography .................................. .. . ......... ................................ ....... 27 iv Daniel Hu A. Problem Under Investigation. of biological processes within an organism The immune system is the collection of identifying and killing pathogens. Defense that protects and defends against disease by identifying Defense against microbes is first mediated of innate immunity and then the later mediated by early reactions of immunity.i Many of immunity such as responses of of adaptive immunity. of the cells involved involved in innate immunity 1 macrophages and neutrophils are involved in phagocytosis of of foreign microbes while recruitment and activation of others such as dendritic cells are involved in recruitment of other cells effective in their role, these cells have a very involved in the immune system. Though effective common among groups of of related limited diversity and are specific specific for structures that are common pathogens. In contrary to the cells involved in the innate immune system, the cells involved in the adaptive immune response such as T cells and B cells have high diversity due to recombination of of gene segments and have extremely high specificity. somatic recombination These cells are involved in cell recruitment, eliminating microbes, and antibody production of T and B among other roles. Adaptive immunity exhibits 'memory' where a portion of cells are maintained maintained as memory cells after after exposure to a pathogen. This allows a much quicker immune response when the same pathogen encountered again. Both innate and pathogen is encountered adaptive immune responses are crucial to the survival of of the organism organism in higher vertebrates and a lack of of these cells can lead to disease. In our study, we focus on of any of of the cells involved in the immune system, particularly particularly the T cell niche. the development development of T cells are responsible for recognizing and eliminating pathogenic invasions in an differentiation organism and, like all blood cells, originate from HSCs. HSCs. 2 Earlier HSC differentiation 2 ultimately leads to two categories of of cells: red blood cells and white blood cells which l Daniel Hu Hu make up the innate and and adaptive immune systems. One working model of hematopoiesis either erythroid/myeloid progenitor cells suggests that during the the process, HSCs become eitber or lymphoid progenitor Erythrocytes cells (Figure 1). I). Then the the progenitor cells specialize EryttvoicVMyeloid Progenitor Cell the speci fi e further into the specific blood Hematopoietic Stem Cell cell Lymphoid cells, Progenitor Cell types. progenitor for for example, differentiate primarily into differentiate Figure 1: 1: Diagram of Hematopoiesis. ly simplified A dramatical dramatically simplified diagram of hematopoeisis, showing the various HSC lineages. 8 cell B cellss or or T T cells while erythroid and and myeloid progenitor cell cellss can can differentiate differentiate into macrophages and and neutrophils. neutrophils. Our Ourarea area of of interest interest is the mechanisms involved invo lved in in T cell development. We use zebrafish as as our our genetic model to use the the zebrafish to identify identify the the various processes of T cell development. With rapid ex ex utero lIIero development, large clutch sizes, and the ability abi lity to to and the perform zebrafish make an the zebrafish an excellent excell ent genetic model to study perfonn forward genetic screens, the developmental Zebrafisb are vertebrates and and the developmental processes. Zebrafish the immune immune system system is is conserved conserved between the zebrafish and and humans, the the exact same cell types in in the zebrafish the two two species sharing the the immune system. Therefore, understanding hematopoiesis in one one species gives great the other.) insight into the other. Anotber zebrafish is that the tbe embryos are are Another advantage of zebrafish transparent. Therefore, it is possible to to view the cells, tissues, and and organs inside the the live li ve can then be different ages as as animal. Physiological comparisons can be made between fish of different 2 Daniel Hu well as between various mutant strains. strains. Just like any other tissue, blood cells begin to form during embryogenesis and therefore, therefore, T cell development can be traced as the fish embryo matures. In zebrafish, the intermediate cell mass of of Oellacher (ICM) is the first place where markers of of hematopoietic stem cells are expressed. expressed.4 The ICM first forms at twelve hours 4 of ventral mesoderm, eventually forming post fertilization and is derived from a subset of the posterior lateral mesoderm. It is unclear if if this first "wave" of of HSPCs contributes to definitive hematopoiesis. The first HSPCs that have been demonstrated to contribute to definitive hematopoietic lineages (e.g. lymphocytes) are found in the dorsal (DA/PCV), and are considered the second ''wave'. wave'. When they aorta/posterior caudal vein (DAlPCV), reach about two days post fertilization (dpf), (dpf). they begin to migrate to the caudal hematopoietic tissue (CHT), and are considered the third 'wave' ' wave' (Figure 2). 2). 5 5 Figure 2: The Location of HSPCs HSpes by Age and Region of of Zebrafish. of HSPCs in the zebrafish zebrafish as well as A diagram showing the spatial and temporal location of noting the analogous human/mouse tissues for each HSPC region. HSPCs are first found in the DAlPCV DA/PCV or aorta/gonad mesonephros (AGM) region in human as highlighted in green. After around 2 dpf, (fetal liver dpf, they begin to migrate and renew in the CHT region (fetal in human) as highlighted in red. Finally, after after 5 dpf, dpf, HSPCs begin to migrate and renew in the kjdney kidney of the fish (bone marrow in human) as highlighted in yellow. Each 'wave' overlaps between ~tween the two regions. 33 Daniel Hu Hu As some of of the HSCs begin to differentiate differentiate into progenitors and more differentiated cells, they travel to their definitive defmitive niche where they are fated to go, such as differentiated T cells to the thymus organ. Subsequently, HSCs migrate to the kidney, their final final destination, where they are produced for the rest of of the animal animal's ' s lifespan. These four destination. main locations are analogous to the yolk sac, aorta/gonad mesonephros, fetal liver, and bone marrow in humans (Figure 2, Section A). For convenience, we will describe HSCs found in the PA/DCV PNDCV as early HSCs and the HSCs found in the CHT as late HSCs. For the T cell niche, the HSCs differentiate differentiate into T cell progenitors, which migrate from the CHT to the thymus of of the zebrafish (Figure 3) around 5 dpf. Once they enter the thymus, progenitors who receive a I ISC ii > progenitors tum into Notch 1I signal turn i m mat urc T Cell immature T cells, which Figure 3: A Diagram of T cell Development. A dramatically simplified diagram of T cell developmentof the various lineages HSCs can undergo. one of eventually become mature T cells. The in situ hybridization technique allows us to label genes that are expressed in different cell types at different different stages of of T cell development. By staining different different different cell types involved in hematopoiesis, it is possible to compare any differences differences in the quantity of cell types between wild-type and mutant individuals. Changes in gene expression of levels and patterns tell us what cell types are affected affected at what point of of T cell development. TC4. Both mutations Our two zebrafish zebrafish mutants that lack T cells are ceylon and TC4. are recessive so in a cross between two heterzygotes for the mutation, we expect twenty- five percent of of the clutch to be mutant. The parents have fluorescently marked T cells so A4 Daniel Hu Hu we can screen the clutch for GFP labeled T cells for mutant fish with reduced fluorescence in the thymus area. Since T cells do not start maturing in the thymus until 6 dpf, dpf, we do not screen until then. However, ceylon ceylon mutants have a morphological jaw defect defect and reduced eye size in addition to reduced T cells so it is possible to screen morphologically normal. Because mutants at younger ages. TC4, on the other hand, is morphologically compromised and they die at an both of of our mutants lack T cells, their immune system is compromised early age. Ceylon Ceylon mutants die at 7 dpf dpf while TC4 mutants die at 16 dpf. We plan to situ hybridization hybridization technique to examine different utilize the in situ different cell types at different different stages of of T cell development defect first occurs in development and determine where the defect hematopoiesis. Analyzing T cell formation in zebrafish zebrafish mutants will allow us to better mechanism of of their development, which will lead to new insights to help understand the mechanism treat T cell related illnesses. B l . Methods (Screening). Bl. ceylon and TC4 are zebrafish Both ceylon zebrafish mutants that lack T cells and therefore, have a defective immune system because the animal does not have the proper means to defend defective defend against pathogens. pathogens. 6 6 identified by crossing ENUThe ceylon ceylon mutants was first identified mutagenized of the WIK background background to wild-type WIK females. mutagenized male zebrafish zebrafish of mutagenesis frequency frequency of of around 1:1000 1: 1000 was determined by A pigment noncomplementation fertilized vitro fertilized noncomplementation tests. Eggs from from heterozygous Fl F 1 females were in vitro with UV inactivated subjected to early pressure in order to duplicate the inactivated sperm and subjected maternal chromosomes chromosomes and generate gynogenetic diploids. diploids. 7 The gynogenetic diploid 7 offspring situ hybridization offspring were grown to 6 dpf dpf and then we performed performed wholemount in situ to gene expression, expression, which labels immature to detect detect rag rag gene which labels immature T T cells cells (Figure (Figure 4). 4). 5 Rag or Rag or Daniel Hu recombination activating gene is an endonuclease necessary for somatic recombination in maturation. T cell maturation. -... . vrtKMype ~ females 'emal~ squeeze 1Q11' eggs IqUMM x I ! We set up a few additional crosses mutagenue males from the F2 map-cross generation to ensure the phenotypes are based on recessive Fl F I to~los rcmalca Ij FI females mutations in the germline of Fl females In vitro fertilize rt'rtJliu ,d lh UV ptnll with 1 V lustllvalt'd inactivated ssperm facts from from earl rather than any arti artifacts earlyy pressure I\ Early .... Karl) PrHSU Pressure ,., mounl lntlon r*9 wholt' wIH-U mount In in sllu situ hybrld hybridization hybridi zation procedures. or RNA in silu situ hybridization FI females are outcrossed to wildFirst, Fl Figure 4: A Diagram of Cross Obtaining Mutants. The in vitro vitro fertilization of of the FI Fl females with UV inactivated spenn sperm and earl create earlyy pressure technique duplicate gynogenetic diploids with duplicate maternal chromosomes. We use in situ situ hybridization to screen for fish with decrease staining of T cells, labeled by rag. 7 type WLK WIK males and the offspring, offspring, once once rai sed to maturity, are then incrossed to raised identify identify heterozygote carriers. Next, the F2 heterozygotes are incrossed and T cells 7 situ hybridization are detected by RNA in situ once more to detect rag gene expression in the F3 offspring. A similar cross was used to identify TC4, TC4, but the TC4 TC4 fish /c&::GFP transgene, which labels immature identify fi sh harbored an Ick::OFP GFP. Rather than identification identification with RNA in situ situ hybridization, and mature T cells with OFP. we identified identified them with a live screen under fluorescence dpf where the fluorescence microscopy at 6 dpf TC4 fish fluorescently labeled T cells when compared to TC4 fi sh would have a severe decrease in fluorescently the wild-type. 82. Methods (Wholemount mRNA In situ B2. situ Hybridization). By using wholemount RNA in situ situ hybridization, we are able to stain various cells of s. of interest and compare the T cell lineage in wild-type and mutant animal animals. 66 This Daniel Hu Daniel Hu technique involves the targeting the lineage-specific lineage-specific mRNA product of of genes that are involved in hematopoiesis and T cell formation. Gene expression expression is differentially differentially regulated during hematopoiesis and T cell development. Therefore, looking at the expression patterns of of specific specific genes during these processes allows us to detect changes of these genes in wild-type and mutant cell in the levels, timing, and patterns of development. We looked at gene expression that is selective to HSPCs, T cell progenitors, and immature and mature T cells to determine defects defects in T cell development ceylon and TC4. Generally, the darker or more abundant the stain, the more cells that in ceylon if a given mutant has a defect defect in those specific are present, so we can determine if specific tissues if if we see a severe decrease of of staining in the mutant compared to the wild-type. situ hybridization hybridization involves generation of The mechanism mechanism of of mRNA in situ of labeled hybridization of of the anti-sense RNA to its target, and detection of anti-sense RNA, hybridization of the mRNA.8 bound mRNA. 8 Anti-sense RNA labeled with an epitope is generated against the sequence for the gene of of interest by in vitro vitro transcription. The anti-sense RNA "probe" is then incubated with the embryos and allowed to bind the target of of interest. The epitope is detected with an anti-epitope antibody conjugated conjugated to an enzyme that facilitates the of a staining agent. Therefore, when the staining substrates are added, they react reaction of with the enzyme and label the cells expressing the gene of of interest. We can then compare different different stages of of T cell development development in the wild-type and mutant strain to determine where the defect defect in development development occurred. Fluorescent mRNA In Situ Situ Hybridization). B3. Methods (Double Fluorescent of the genes we are looking at Because some cell types express more than one of of expression of of one gene than the other, it is advantageous to there may be a greater loss of 7 Daniel Daniel Hu look at at both both gene gene expression expression in the the same same animal animal since since there there is variance variance between between different look different zebrafish. For For example, HSPCs HSPCs express express both both cmyb cmyb and and ikaros ikaros genes, but but we we don't don't know zebrafish. if the expression expression of of both both genes genes is reduced reduced to the the same same level level in in the mutant mutant and and if if it is if reduced globally globally or in single cells. A A greater greater reduction reduction of of ikaros ikaros than than cmyb cmyb means the reduced defect is different different than than if if both genes expressions expressions are reduced reduced equally. Also, some subsets defect of HSPCs HSPCs are identifiable identifiable by coexpression coexpression of of genes. For For example, it may may be possible possible that of HSPCs only express express cmyb, cmyb, some only ikaros, ikaros, and and some some express express both. some HSPCs By identifying which populations populations of of cells are affected, affected, we will have a greater understanding identifying of how the mutation mutation affects affects hematopoiesis. of fluorescent in situ situ is similar similar to the standard standard mRNA mRNA in The procedure for double fluorescent situ hybridization hybridization protocol. To look at the expression expression of of two genes at the same time, we situ make one anti-sense RNA probe for each gene, but one probe will contain a digoxygenin and the other a fluorescein important that the epitope conjugated conjugated to the probes fluorescein tag. It is important different so each signal can be detected separately. Preferably, are different Preferably, the weaker weaker probe (or fluorescein the probe to the gene expressed at lower levels) should be labeled with fluorescein because it amplifies amplifies the signal better when labeled fluorescently. Both probes are added to the embryos at the same time during the hybridization step. As in the standard RNA in situ situ hybridization protocol, either anti-digoxygenin or anti-fluorescein is added after after the hybridization step to detect the anti-sense probe. In anti-fluorescein contrast to the standard RNA in situ situ hybridization protocol, a peroxidase enzyme is conjugated with the antibody to react with the fluorescent staining agent. We have found found conjugated adding the anti-fluorescein anti-fluorescein first results in non-specific double labeling of of cells and therefore, the anti-digoxygenin is added first. The two different different fluorescent peroxidase peroxidase 88 Daniel Hu Daniel Hu substrates substrates we we use are tyramide-FITC, which which emits emits at 520 nm nm and and tyramide-cy3, which emits at 570 nm. Cy3 is a weaker weaker label label so we add add it with with our our stronger stronger probe. After After the emits first antibody antibody is detected, a deactivation deactivation process process inactivates inactivates the peroxidase peroxidase from from the first first antibody. embryos are incubated incubated with the second second antibody antibody and and a second Then, the embryos labeling reaction reaction is performed. labeling several advantages advantages and and disadvantages disadvantages of of fluorescent fluorescent labeling Overall, there are several previously described described chromogenic chromogenic detection detection method method using using NBT/BCIP versus the previously staining. The problem problem with fluorescence fluorescence is that it is light sensitive, cannot be seen without fluorescent fluorescent lighting, and have less amplification amplification of of the signal. This can result in a failure to see a weaker probe. However, fluorescence signaling creates much better of double fluorescent fluorescent in situs situs is that the labeling spatial resolution. The main advantage of procedure does not form a precipitate as occurs in the chromogenic method. The problem problem with the precipitate precipitate is that the darker precipitate blocks the fluorescent fluorescent staining or even a lighter precipitate no matter how underexpressed underexpressed the darker stain or overexpressed fluorescent/lighter stain. As a result, it makes detecting coexpression overexpressed the fluorescent/lighter difficult fluorescent in situ difficult and therefore, we favor the double fluorescent situ hybridization procedure. C. Current Results. We asked five central questions about our ceylon ceylon and TC4 mutant phenotypes. if ceylon ceylon and TC4 lack all types of of T cells, resulting in their First, we checked if immunodeficiency. The genes rag rag and lck Ick are expressed in immature and mature T cells immunodeficiency. 9 respectively. Similarly, tdt labels T cells that are undergoing somatic recombination, recombination, respectively.9 which is part of of the T cell maturation process. Second, we asked if the other cell types in the thymus are affected affected in mutant embryos. We address this question by looking at the 9 Daniel Hu expression of of the gene foxnl foxnl in thymic epithelia cells, which are essential for thymus if T cell progenitors are affected affected in the mutant embryo by formation. Third, we tested if using cmyb cmyb and ikaros ikaros probes. Fourth, we asked what stage of affected of hematopoiesis is affected of in the mutant embryos. To address this question, we looked at the expression expression pattern of different genes expressed in HSPCs at multiple developmental time points several different (Figure 5). Finally, we decided to look at other HSPC lineages to see if if other blood cell mpo gene is expressed in neutrophils, a cell type in the innate types are affected. affected. The mpo Probe Cell Expressed/Stained Expressed/Stained Cell Location Embryo Age rag rag Immature T cells Thymus (Head) 6 dpf lck Ick Maturing and mature T cells Thymus (Head) 6 dpf tdt T cells undergoing somatic recombination recombination Thymus (Head) 6 dpf foxnl foxnl Thymic epithelial cells Thymus (Head) 6 dpf ikaros ikaros T cell progenitors Thymus (Head) 6 dpf cmyb cmyb T cell progenitors, Late HSPCs and Early HSPCs Thymus-T cell progenitors (Head) CHT-Late HSPCs DA/PCV-Early HSPCs DA/PCV-Early 6 ddpf p f ((T T cell progenitors, Late HSPCs) 2 dpf dpf (early HSPCs) Imo2 lmo2 Late HSPCs CHT -Late HSPCs CHT-Late 6 dpf dpf scl scl Late HSPCs HSPCs mpo mpo Neutrophils and Early CHT-Late HSPCs DA/PCV-Early HSPCs DA/PCV-Early CHT and AGM 6 dpf dpf (late HSPCs) 1I dpf (early (early HSPCs) HSPCs) 6 dpf Figure 5: Anti-Sense RNA Probes and the Cells that are Labeled. A chart designating the probes used, the cells labeled by each probe, the location of of the experiment was performed. cells in relation to the fish embryo, and the embryo's age the experiment performed. 10 Daniel Hu immune system and derived from myeloid progenitors. progenitors. IO As explained in Section B2, 10 we used wholemount in situ situ hybridization to label the cells that express the specific gene and compare the staining between wild-type and mutant animals. situ hybridization We analyze and image the RNA in situ hybridization with a camera attached to a dissecting microscope or compound microscope. These analyses will allow us to identify identify the specific specific times and cell types affected affected during hematopoiesis in the T cell defective defective mutants. In the following figures, we have shown a side-by-side comparison of of ceylon ceylon and TC4 mutants labeled with the same probe and each with its own wild-type control. Since experiments were performed ceylon and TC4, a wild-type performed at different different times with ceylon control will be depicted for each mutant. The first question we addressed was whether both immature and mature T cells are affected ceylon mutant. To address this question, we examined the expression affected in the ceylon level of rag, which is expressed in immature T cells, lck, of rag, Ick, which is expressed expressed in maturing and mature T cells, and tdt, which is expressed in T cells undergoing somatic Ceylon and its respective wildrecombination as described above (Figure 5, Section C). Ceylon dpf while TC4 and its type control was stained separately with all three probes at 6 dpf respective wild-type control was stained with only rag Ick. rag and lck. As indicated by the arrow, the staining in the thymus region where the T cells are located is prominent in wild-type for each probe, but is much smaller in both the ceylon ceylon and TC4 mutant (Figure 6). These results suggest that both immature T cells and mature T cells in ceylon ceylon and TC4 are reduced. TC4 are reduced. 11 Daniel Hu WT ceylon WT 7C4 Figure 6: Both immature and mature T cells are reduced in ceylon and TC4 mutants. (a-j) 6 dpf embryos, anterior to the left, red arrow indicates T cell location, location. rag expression is reduced in the ceylon mutant (b) as compared to wild-type (a). Ick expression is missing in the ceylon mutant (f) (t) as compared to wild-type wi ld-type (e). Ceylon mutants have reduced tdt expression (j) Q) as compared to wild-type (i). (i). rag expression, shown in fluorescence, is reduced in the TC4 mutant (d) as compared to wild-type (c). (c). Ick expression is missing in the TC4 mutant (h) as compared to wildtype (g). Now that we know that T cells are either reduced or missing in both ceylon and TC4 affected. TC4 mutants, we asked our second question of of whether thymus development is affected. dpf To answer this thi s question, we looked at wild-type, ceylon, and TC4 animals fixed at 6 dpf foxnl expression, which labels the thymic epithelial cells and are crucial in forming for foxnl the thymus. The staining in the thymus region of of ceylon is nearly the same size as wildtype (Figure 7). 7). However, thymic epithelial staining in the thymus of of TC4 is nearly gone. 12 Daniel Hu These results show that the thymus of the ceylon mutant is only slightly smaller if at all when compared to the wild-type, but the thymus of of the TC4 TC4 mutant is nearly gone. gone. WT ceylon WT ~- fOIlnf TEC. Up' nea rly normal in ceylon mutants, Figure 7: Thymic epithelial cells (TEes) (TECs) are nearly but severely reduced in TC4 TC4 mutants. (a-d) 6 dpf dpf embryos, anterior to the left, red arrow indicates thymic epithelial cell (TEe) foxn I expression in the ceylon mutant (b) is about the same size as (TEC) location. location, foxnl foxnl expression on the wild-type (a). The darker stain above the red arrow is the foxnl (a). other side of the fish. However, TC4 TC4 mutants (d) have a reduced thymus size compared to the wild-type (c). if the the T progenitors that become T We next asked We next asked if T cell cell progenitors that eventually eventually become T cells cells are are affected affected in the the ceylon probes, cymb in ceylon and and TC4 TC4 mutant. mutant. We We stained stained ceylon ceylon separately separately with with two two probes, cymb and and ikaros, that are for T progenitors, located the thymus. thymus. TC4 was stained ikaros, that are specific specific for T cell cell progenitors, located in in the TC4 was stained only with ikaros. We examined progenitors in in wild-type, wi ld-type, ceylon, only with ikaros. We examined T T cell cell progenitors ceylon, and and TC4 TC4 animals animals at the red red arrow, is reduced reduced in in ceylon TC4 at 66 dpf. dpf. The The staining, staining, as as designated designated by by the arrow, is ceylon and and TC4 compared to the ld-type (Figure results suggest progenitors that compared to the wi wild-type (Figure 8). 8). These These results suggest that that T T cell cell progenitors that express reduced in both ceylon express ikaros ikaros and/or and/or cmyb cmyb are are reduced in both ceylon and and TC4 TC4 mutants. mutants. 13 Daniel Hu WT WT ceylon TC4 ikaros HSPCs 6dpf Gdpf i i cmyb cmyb HSPCs 6dpf 8dpf mutants.. Figure 8: T cell progenitors are reduced in ceylon and TC4 mutants (a-f) 6 dpf embryos, anterior to the left, red arrow indicates T cell progenitor location. ikaros expression in the ceylon mutant (b) is reduced compared to wild-type (a), (a). cmyb expression in the ceylon mutant (f) is reduced compared to wild-type wi ld-type (e). ikaros expression, shown in fluorescence, in the TC4 mutant (d) is reduced compared to wildtype (c). (e). For our fourth question, we addressed at what point in development if if at all are HSPCs present in ceylon and TC4 TC4 mutants. Late HSPCs are located in the CHT region of of Imo2 probes to label late HSPCs in ceylon the fish at 6 dpf. We used both sci scl and lmo2 controll at 6 dpf. For TC4 and its wild-type animals and its wild-type contro wi ld-type control, we used cmyb to stain late HSPCs at 6 dpf. We noticed that the staining in the tail is smaller in TC4 compared to the wild-type (Figure 9). These results show that late ceylon and TC4 HSPCs are reduced in ceylon and TC4 TC4 mutants. 14 Daniel Daniel Hu .., c.ylon WT • • I HS"" I Up' ._. H$"" • • I I Up, Figure 9: 9: Late "Spes HSPCs are reduced in ceylon and TC4 mutants. (a-f) 6 dpf embryos, posterior to the right, red arrow indicates late HSPC location, location. sci scl expression in the ceylon mutant (b) is reduced compared to wild-type (a). lmo2 fmo2 expression in the ceylon mutant (t) (f) is reduced compared to wild-type (e). cmyb expression, shown in fluorescence, fluorescence, in the TC4 mutant (d) is reduced compared to wildtype (e). (c). After looking at late HSPCs in 6dpf 6dpf embryos, we looked at early HSPCs in 1-2dpf l-2dpf of the embryos. At this stage, the HSPCs are found both in the AGM and CHT region of fish. To address this issue, we looked at scl sci expression at 1 dpf dpfand and cmyb expression at 2 dpfin dpf in wild-type and ceylon. Both of of these genes are expressed in early HSPCs. We have not yet looked at early HSPCs in TC4 mutants. When we examine the staining in the tail, we see nearly the same amount of wi ld-type and ceylon animals of stained cells between the wild-type nonna! in ceylon mutants (Figure 10). We find from these results that early HSPCs are normal despite the fact late HSPCs are defective. 15 15 Daniel Hu ceylon ceylon WT sci scl • bb • HSPO 1dpf 1dpf cmyb cmyb HSPC HSPC 2dpf 2dpf Ea rly HSpes Figure 10: Early HSPCs are normal in ceylon mutants. (a-b) 1I dpf embryos, posterior to the right, early HSPC location shown throughout tail. sci wi ld-type (a) is normal, normal. tail, scl expression in both the ceylon mutant (b) and wild-type (c-d) 2 dpf embryos, posterior to the right, early HSPC location shown throughout tail. cmyb expression in both the ceylon mutant (d) and wild-type wi ld-type (c) is normal. tail, Our results suggest that early HSPCs form normally in ceylon mutants, but the later stages of hematopoiesis of the T cell lineage is defective. To determine whether defects are specific to the T cell lineage, we analyzed other blood lineages. We examined expression of mpo, a marker for neutrophils, which derive from the myeloid lineage and are found all over the fish. We stained wi ld-type, ceylon, and TC4 wild-type, TC4 fish at 6 dpf and looked specifically specifically at the tail. We see nearly the same amount of stained cells between the wild-type and ceylon animals though TC4 I I). These TC4 stain is much smaller (Figure 11). results show that neutrophils in ceylon mutants, just like with early HSPCs, are also normaJ, but neutrophils in TC4 mutants are reduced. normal, 16 Daniel Hu Daniel cmyton .,."... WT -........ ....' • • , I WT 0 • • • ~ TC4 I • t 1: Neutrophils (PMNs) are normal in ceylon mutants, but reduced in TC4 Figure 11: mutants. (a-d) 6 dpf location. mpo dpf embryos, posterior to the right, red arrow indicates neutrophil location, expression in the ceylon mutant (b) and wild-type (a) is normal, normal. mpo expression in the TC4 TC4 mutant (d) is reduced compared to wild-type (c). Outside of the five questions we asked earlier (Section B2), we also are interested in seeing if there is any coexpression of if so, is there a greater of rag and Ick in T cells and if lck. We have yet to perform the decrease of of T cells in ceylon that coexpress rag and Ick. double in situ experiment on TC4. TC4. We have recently finished troubleshooting the double fluorescent in situ hybridization protocol. So far, we have achieved satisfactory satisfactory results ragllck, rag/cmyb, raglcmyb, and Icklcmyb in wild-type animals (Figure 12). with combinations of of rag/lck, The rag expressing cells in the thymus (immature T cells) are shown in green while the cmyb expressed cells in the thymus (T-cell progenitors) are shown in red. Cells that coexpress the two genes are stained in yellow. The fact that we see staining of of two different different sets of celJs cells along with some overlap from coexpression shows that we have a protocoL working double fluorescent in situ protocol. t7 17 Daniel Hu Figure 12: Double Fluorescent In Situs on Wild Type Animals. (a-c) 6 dpf dpf wild type embryos taken at 40x with confocal microscopy, anterior to the left, showing cells that are found in the thymus. Figure 12a is a double in situ of left, of ragllck. showing immature T cells (rag) in green and mature T cells (lek) ragllck, (Ick) in red. Figure 12b is a double in situ of of raglcmyb, rag/cmyb, showing immature T cells in green (rag) and T cell progenitors in red (emyb). l2c is a double in situ of (cmyb). Figure 12c of Ick/cmyb Ick/cmyb,, showing mature T cells (lek) (Ick) in green and T cell progenitors in red (emyb). (cmyb). Cells that coexpress the two gene expressions are in yellow. In the double fluorescent for ceylon and the wild-type control at 6 dpf, we stained In the double fluorescent for ceylon and the wild-type control at 6 dpf, we stained rag-expressed cells (immature T cells) in red and Ick-expressed cells (mature T cells) in rag-expressed cells (immature T cells) in red and /cA>expressed cells (mature T cells) in green. Both of these cells are found in the thymus region. In the wild-type, we found green. Both of these cells are found in the thymus region. In the wild-type, we found that there are only low amounts of T cells that coexpress rag and lek. As predicted, both that there are only low amounts of T cells that coexpress rag and Ick. As predicted, both immature and mature T cells are severely reduced in the ceylon mutant when compared to immature and mature T cells are severely reduced in the ceylon mutant when compared to the wild-type fish (Figure 13). Also. there seems to be a much higher number of the wild-type fish (Figure 13). Also, there seems to be a much higher number of immature T cells than mature T cells in ceylon, which is consistent with our previous data immature T cells than mature T cells in ceylon, which is consistent with our previous data (Figure 6, Section C). In addition, out of the few T cells that are present in ceylon, none (Figure 6, Section C). In addition, out of the few T cells that are present in ceylon, none of them show coexpression. of them show coexpression. 18 Daniel Hu ceylon WT Figure 13: 13: T cells in ceylon do not show coexpression. confocal microscopy, (a-b) 6 dpf dpf wild type embryos taken at 40x with confocal anterior to the left, showing cells that are found in the thymus. Rag labeled cells are shown in red, lck Ick labeled cells are shown in green, and cells coexpresing rag and Ick are shown in yellow. Rag/lck expression in the lck ceylon mutant (b) is severely reduced compared to wild-type (a). Also Ick is much more reduced compared to rag in ceylon. Finally, T cells in ceylon do not show coexpression while some T cells in wild-type do. Now that we have examined which cells involved in hematopoiesis are defective, we want to examine the morphological defects in ceylon. Though the TC4 mutant is normal , the ceylon mutant exhibits a jaw defect and reduced eye size. morphologically normal, Since the cartilage carti lage tissue ti ssue in the jaw derives from the endoderm, we wanted to see if if the ceylon mutant has defects in any other endoderm-derived tissues. To determine this, we performed in situ hybridization experiments with lineage-specific lineage-specific anti-sense RNA probes that label other cell types including endoderm-derived tissues, which is also the main 11 germ layer responsible for the formation of of the thymic epithelial cells of of the thymus. thymus.I I gata6, and trypsin as well as Alcian blue So far, we have used cmcl2, tbxl, tbxi , gata6, staining to target the heart, ear, gut, pancreas, and cartilage growth respectively in six day embryos (Figure 14). 12 Similarly to the in situ hybridizations with the T cell lineage, these probes were run using the same conditions as those experiments. Designated by the 19 Daniel Hu Daniel wild·type and ceylon red arrow, staining in the ear and heart is about the same between wild-type ceylon animals. However, However, staini ng in the gut and pancreas is much smaller staining smaller in the ceylon ceylon mutant compared to wild-type. compared wild·type. Furthermore, carti cartilage staining ceylon mutants is very lage sta ining in ceylon disrupted compared to wild-type. Out of of these tissues, the gut, pancreas, and of all of cartilage show severe disruption and size decrease while whi le the heart and the ear appear normal (Figure 15). Probe Cell Cellss Labeled Embryo Age cmel2 cmcl2 Heart 66dpf dpf tbxl tbxl Ear 66dpf dpf gafa gata66 Gut 66dpf dpf trypsin trypsin Pancreas 6 ddpf pf Alcian blue Cartilage 6 ddpf pf Anti-Sense RNA Probes and the Tissues that are Labeled. Figure 14: Anti-Sense ' s age the A chart designating the probes used, what cells are labeled and the embryo embryo's experiment was performed. performed. 20 Daniel Hu wr _ .., WT ..... . ...... .... <0<_ • ... - tid.' .... JO t • • .a. ..", tbxi Sdpf U pl Figure 15: Some endoderm-derived tissues are reduced in ceylon ceylon mutants. (a-j) 6 dpf embryos, posterior to the right, red arrow indicates tissue location. Alcian Blue is a molecule that stains cartilage and shows that cartilage is disrupted in the ceylon mutant (b) and not in wild-type (a). (a). gala6, gata6, expressed in the gut, is reduced in the ceylon mutant (d) when compared to the wild-type (c). (c). trypsin, expressed in the pancreas, is reduced in the ceylon mutant (f) when compared to the wild-type (e). cmctt, expressed in the heart, is normal cmel2, nonnal in both ceylon mutants (h) and wild-type (g). tbxl, (i). tbxl, expressed in the ear, is normal in both ceylon mutants (j) and wild-type (i). We have identified a deletion on the telomere of chromosome 3 where the ceylon LG3 \6). Since there are a multiple genes in this mutant maps (Figure 16). region, we were curious to detennine determine if if one of the genes had a predominant effect effect on the ceylon phenotype. Z8681Z1191Z53495Z25883- Z1401Z61617- •foxjl •sox9b •zgc:152997 •sox8 -zgc:113100 The tbl3 Ib/3 gene is expressed in gut, pancreas region, neural crest, eye, and late HSPC Figure 16: Deletion Where Ceylon Ceylon Mutant Maps. Region showing all the known genes deleted in the ceylon mutant as listed li sted on the right ri ght hand side and all the known markers deleted in the ceylon mutant as listed on the left hand side on linkage group (chromosome) 3. •tbl3 noxol 21 21 Hu Daniel Hu region. 13 Since these regions are all defective in the ceylon region. ceylon mutant, we hypothesized that 13 of the late hematopoietic disruption in ceylon the loss tbl3 is a major major cause of ceylon that results in a lack of of T cells. To test our hypothesis, we injected injected antisense DNA oligonucleotides oligonucleotides fbl3 into wild-type animals. direction against tbl3 Our results revealed that the injected injected animals phenocopied ceylon, ceylon, which supports our hypothesis. D. Discussion. In summary, mutant lacks progenitors, immature, In summary, the the ceylon ceylon mutant lacks late late HSPCs, HSPCs, T T cell cell progenitors, immature, and mature T HSPCs, neutrophils, and mature T cells. cells. However, However, early early HSPCs, neutrophils, and and thymic thymic epithelial epithelial cells cells are are mutants compared wild-type. Our nearly normal nearly normal in in ceylon ceylon mutants compared to to wild-type. Our double double fluorescent fluorescent in situ situ showed that there be aa much much higher number of showed that there seems seems to to be higher number of immature immature T T cells cells than than mature mature T T ceylon mutants, mutants, suggesting the T cell lineage cells in ceylon cells in suggesting cells cells further further down down the T cell lineage are are more more In other we noted noted that gut, and disrupted than ones disrupted than ones earlier. earlier. In other tissues, tissues, we that cartilage, cartilage, gut, and pancreas pancreas were either Finally, we we were were able by injecting were either disrupted disrupted or or reduced. reduced. Finally, able to to phenocopy phenocopy ceylon ceylon by injecting antisense DNA against tbl3, fbl3, a gene that is part of of the deletion where ceylon ceylon maps, into fbl3 is expressed in gut, pancreas region, neural crest, eye, and late wild-type fish. Since tbl3 HSPC region, which are all defective in the ceylon ceylon mutant, it is likely that tbl3 is a major major cause of of the ceylon ceylon phenotypes. Because the T cell lineage in the ceylon ceylon mutant is defective, but neutrophils are normal, the myeloid lineage where the neutrophils derive from may not be affected affected by the mutation despite a lack of of late HSPCs. Because early HSPCs are normal, it could mean that neutrophils are primarily made by the early HSPCs. Another suggestion is that the defect in the renewal process. HSPCs are still making neutrophils but there is a defect 22 22 Daniel Hu Hu Another interesting result to note is that, despite a lack of of T cells, the thymus is mutant compared compared to wild-type. Since thymic epithelial cell nearly normal in the ceylon ceylon mutant development is interdependent interdependent with T cell development, a lack of of T cells might result in a of thymus. thymus. 14 The fact that the thymus in the ceylon ceylon mutant lack or marked reduction of 14 looks almost normal when it should not due to a severe reduction of of T cells suggest that development of there could be a problem in the development of thymic epithelial cells. Therefore, it is development such as CHT and the thymus, possible that the tissues supporting late HSPC development supporting T cell development, are both defective. However, based on our results with fbl3, it is is most most likely that the the mutation mutation in in ceylon results in tbl3, it likely that ceylon results in aa hematopoietic-intrinsic hematopoietic-intrinsic of differentiation differentiation into into the the lymphoid disruption disruption of lymphoid lineage. lineage. makes it an interesting gene gene to to study. From Not much much is known known about about fbl3, Not tbl3, which makes of tryptophan-aspartate the little we know, fbl3 tbl3 codes for a protein that contains a series of tryptophan-aspartate repeats. Such proteins are involved in protein-protein protein-protein interactions in transcription transcription factors. regulations, forming scaffolds scaffolds with transcription Two proteins in the same family as Tbl3, Tb13, Tbll and TblRl, have been confirmed confirmed for those roles. We want to focus family focus of fbl3 understand the mechanism mechanism behind effects effects caused by the our studies on the role of tbl3 to understand loss of of the gene in ceylon. ceylon. Similarly to the ceylon ceylon mutant, TC4 mutants are also missing late HSPCs, T cell progenitors, immature, and mature T cells. In contrast, neutrophils and thymic epithelial of HSPCs further further suggests that the cells are reduced reduced in TC4 mutants. Severe reduction of of all lymphoid lymphoid lineages, B cells, could be affected affected as well, resulting in a loss of cells. Because of of the interdependent interdependent relation between between the thymus and T cells, the reduction of of thymic epithelial cells in TC4 is expected expected because there is a reduction of of T 23 Daniel Hu Daniel Hu because neutrophils neutrophils are barely barely detectable detectable in the TC4 TC4 embryos, we know cells. Finally, because that non-lymphoid non-lymphoid blood blood lineages lineages such such as the myeloid myeloid lineage, are also affected. affected. Based that on these results, we have have deduced deduced that that the problem problem occurs occurs very very early early in hematopoiesis, on of the TC4 defects in both the lymphoid lymphoid and erythroid/myeloid erythroid/myeloid lineages of which leads to defects ceylon mutants, there are no other other morphological morphological defects defects in mutant. Since, unlike with ceylon that the TC4 mutation mutation is blood-lineage blood-lineage specific. the TC4, it is likely that E. Future Directions. Our goal goal in in analyzing analyzing ceylon ceylon and and TC4 mutants mutants is is to to determine determine the the defects in T T cell Our defects in cell of T T cells cells cause cause immune development that that the the mutations mutations cause. cause. Since reduction or or loss loss of development Since aa reduction immune system related related diseases diseases such as immunodeficiency, immunodeficiency, these these mutants mutants will will be be used used to to gain system such as gain aa better understanding understanding of of the the developmental developmental processes processes of of T T cells. cells. There There are are two important better be taken taken for for the the ceylon ceylon project project to to better better support our our hypothesis hypothesis that that the the fb13 steps that that will will be tbl3 is the predomiant predomiant cause of ceylon. If of the defects in ceylon. If this is the case, we can focus our studies on the role of of tbl3, fbl3, determining its function function in late intrinsic HSPCs signaling to better achieve our goal. First, we intend to inject inject wild tlb3 flb3 into the ceylon ceylon mutants to see if if we can rescue T cell and/or organ development. Second, we want to identify identify the different different subtypes subtypes of of blood cells that are affected affected in ceylon ceylon mutants by look at coexpression of of two genes expressed in two cell types simultaneously simultaneously using double fluorescent RNA in situ situ of these hybridizations. This will allow us to analyze populations that express only one of genes and others that express both these genes, allowing us to determine if they are differentially differentially affected affected in the mutants. mutants.IS Once we have colabeled multiple gene 15 expression patterns, we can start a quantitative assay, comparing the exact number of a 24 24 Daniel Hu Daniel Hu specific cell cell type type between between a wild-type wild-type and and mutant mutant zebrafish. zebrafish. specific Specifically in the near Specifically looking at coexpression coexpression of of rag/cmyb rag/cmyb in in immature immature T T cells and and T T cell cell progenitors, future, looking Ick/cmyb in in mature mature T T cells cells and and thymic thymic epithelial epithelial cells, and and rag/foxnl raglfoxnl in in immature immature TT cells Ick/cmyb further our our understanding understanding of of the and thymic epithelial epithelial cells will be important important to further and relationship between between thymus, T cells, and and T cell cell progenitors. relationship TC4 mutant, we want want to analyze coexpression coexpression of of the same Similarly, with the TC4 of rag/lck rag/lck in immature immature and and mature T cells, which which has genes listed listed above with the addition addition of not been been done done on on this this mutant. mutant. We We plan plan both both qualitative qualitative and and quantitative quantitative analyses analyses of of the not the cells coexpressing coexpressing the the genes genes of of interest. interest. cells In addition, addition, analysis analysis of of T cells, T T cell In T cells, cell is important important to to see see at at what what point progenitors, and and especially especially HSPCs HSPCs in in younger younger embryos embryos is progenitors, of development development the defects defects first first occur. Finally, we want to identify identify the genetic lesion that of of that gene in hematopoiesis. With these causes these problems and identify identify the role of ceylon and TC4 and thereby, increase future experiments, we can advance studies of of both ceylon our knowledge about T cell development development to better understand the cause and mechanism ofT of T cell related diseases. F. Conclusion. T cell development IS is an important field of research because T cells are an intricate part of of the adaptive immune system and disruption in the differentiation differentiation of of T cells leads to lethal illnesses including immunodeficiency. immunodeficiency. By using zebrafish zebrafish as a genetic model organism for human diseases, we can study the process of of hematopoiesis hematopoiesis and use mutants to determine how this process is regulated. Our current research has ceylon mutant not only causes disruption in late shown that the deletion of tbl3 fbl3 in the ceylon tbl3, HSPCs that results in lack of T cells, but also various other tissues that express fbl3, 25 Daniel Daniel Hu Hu which could potentially potentiall y cause cause disruption di sruption in in the the tissues tissues that that support support hematopoiesis. which Therefore, we we have opened opened up up an expanding expand ing field field of of possibilities possibiliti es by by identifying ide nti fy ing aa new Therefore, gene involved invo lved in the development development of of the the thymus, T cell ce ll progenitors progenitors and and T cells. gene In our our TC4 TC4 mutant, mutant, we have discovered that the the mutation mutation causes causes a problem problem in In early hematopoiesis hematopoiesis that that results in defects defects in both the the lymphoid lymphoid lineage lineage and the erythroid/myeloid lineages. li neages. Discovering Discovering the lesion lesion in this thi s mutant will contribute contribute to to our erythroid/myeloid of the the signaling signaling involved involved in HSPCs ce in g understand ing of understanding cellll fate determination. Understand Understanding development and the genes involved invo lved in this thi s process will lead to better treatments T cell development for diseases. for immunodeficiency immunodeficiency and other other related di seases. 26 Daniel Hu Daniel Hu G. Bibliography. 1. Silverthorn DU. Human Physiology: An Integrated Integrated Approach 44th ed. San Francisco, CA: Pearson Education, Inc., 2009. chp. 24. l 2. Trede NS, Zapata A, Zon LI. "Fishing for lymphoid lymphoid genes." Trends Immunol. 2001 Jun;22(6):302-7. Langenau DM, OM, Traver D, 0, Look AT, Zon LI. "The use of of zebrafish zebrafish to 3. Trede NS, Langenau understand immunity." Immunity. 2004 Apr;20( 4):367-79. understand Apr;20(4):367-79. OW, Catic A, 4. 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