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The main focus of this paper is to see whether Kitb/kitlgb is necessary for HSC’s in the zebrafish model, if osm is a new cytokine important for HSC’s in the zebrafish model, whether osmr and kitb signalling are required sequentially for HSC specification, and whether osmr and kitb synergize to expand HSC’s in the caudal hematopoietic tissue. These are important because understanding molecular pathways that control the specification and expansion of hematopoietic stem cells is necessary for developing and performing regenerative medication.
Ha: si:ch73-47f2. 1 is the zebrafish ortholog of the mammalian OSM, the gene that encodes for the osm protein Ho: si:ch73-47f2. 1 is not the zebrafish ortholog of the mammalian OSM4. The expression of osm was not detectable by in-situ hybridization (IHS), so cEC’s (caudal embryonic cells) were isolated by FACS (fluorescence activated cell sorting). This is a method for sorting a mixture of cells one at a time into separate containers using electrical charges based on the light scattering and fluorescent properties of each cell. After the cells were isolated they were amplified using qPCR (quantitative polymerase chain reaction). This is done by denaturing a target strand of DNA, annealing it with another strand to isolate the area of interest and elongating it as in normal transcription. The difference is that a fluorescent label is added to quantify the the amplified DNA as it amplifies. The data represented in he figures are averages of biological triplicates meaning the whole procedure was done 3 separate times to control for biological variation.
eGFP+ on the x-axis is an enhanced green fluorescent protein used in the qPCR to detect for the presence of the target protein, in this case, osm on the flk1 gene of the zebrafish. On the y-axis, a fold change simply represents how much the protein expression increased by. A change of 0-50 represented on the y-axis represents a fold change of 2. This means that the gene is expressing twice as much on 100 as it is on 50. In the graph, it is evident that there was more than twice as much expression of osm in the caudal hematopoietic tissue (CHT) or, the tail, compared to the rest of the embryo.
Based on the results on the graph, it is evident that osm is expressed in high volumes in the CHT of the zebrafish model. The expression of osm in zebrafish, as in mammals, indicates a role in hematopoiesis (formation of blood cells), also as in mammals, supporting the hypothesis that si:ch73-47f2. 1 is the ortholog of the mammalian OSM gene that codes for the osm protein involved in hematopoiesis, thus, failing to reject the null hypothesis.
The information shows how osm expands HSC’s within the caudal hematopoietic tissue. It shows that there was a significant increase in the number of HSC’s in the CHT of the embryo’s injected with osm mRNA. Osmr is the receptor that binds to osm to activate it. This helps show that osm and its receptor osmr play a role in the expansion of HSC’s in the CHT. One of the main questions of this paper was to see whether there is an expansion of HSC’s (hematopoietic stem cells) in the caudal hematopoietic tissue (CHT) caused by a synergy between osmr and kitb. This information helps narrow down osmr as a factor of expansion of HSC’s in the CHT. The role of kitb would now have to be analyzed to further help answer the main question.
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