Hämatopoetische Stammzellen: Regulation durch runxa und andere Transkriptionsfaktoren während Homeostase und Differenzierung im Zebrafisch-Modell
Final Report Abstract
Blood and blood vessel formation represent processes essential for normal development as well as for the occurrence of diseases such as cancer. The zebrafish has proven to be especially useful for studying blood development. By characterizing zebrafish mutants with defective blood formation, several genes that serve as master regulators during blood (hematopoietic) and blood vessel (vasculogenetic) formation had previously been identified. My studies analyzed the genetic regulation of blood development by comparing genome wide expression profiles of zebrafish mutants to their wild type siblings. My experiments identified not only the underlying genetic mutations, they also revealed genetic misregulations that occur further downstream as a result of the mutation. These misregulations included known hematopoietic genes as well as genes without previously known function during blood development. Additional microarray experiments on fluorescently labelled blood progenitor populations classified these novel genes further. Their expression could be linked to blood lineage determination as well as to early or late function during development. Several of these stem and progenitor cell genes were expressed at a very early developmental stage, during the primitive wave of hematopoeisis. Genetic regulations during these early stages are of particular interest since they are likely to play an essential role during differentiation of more primitive stem cells into blood stem cells. Abnormal expression of similar early stem cell genes, such as scl, lmo2 or gata) have also been shown to be crucial for specific leukemias to occur. Since the early genes identified in my screen were simultaneously expressed with scl, lmo2 and gata1, they are likely to play similar key regulatory roles during blood development. Probing this very early hematopoietic stage is technically challenging since the cell number at this timepoint is very limited. Studies in mammalian systems have only been capable to analyze later developmental stages. The microarray studies presented here analyzed not only stem cells of this primitive blood wave, but also more primitive hemangioblasts which give rise to blood and blood vessels during regular vertebrate development. These results thereby represent the first large scale study on the genetic regulation of the earliest stages of vertebral blood development. Some of these novel genes were genetically linked to the cloche mutation, cloche mutants are characterized by absent blood and blood vessels, with the exact genetic defect remaining elusive. However, testing these novel genes in loss of function or genetic rescue experiments did not reproduce or rescue the cloche phenotype, respectively. The lack of detecting the mutated gene might be due to incomplete representation of the zebrafish genome on the Affymetrix array. Newer, more complete versions of the array as well new genome DNA arrays will likely solve this problem. The microarray studies presented here analyzed the genetic regulation of blood stem cells in zebrafish. Since the hematopoietic gene program is evolutionarily highly conserved among vertebrates, the insight gained from these studies will have great impact on our understanding of the process of human blood formation.
Publications
- A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc Natl Acad Sci USA . 2005;102(37):13194-9
Shepard JL, Amatruda JF, Stern HM, Subramanian A, Finkelstein D, Ziai J, Finley KR, Pfaff KL, Hersey C, Zhou Y, Barut B, Freedman M, Lee C, Spitsbergen J, Neuberg D, Weber G, Golub TR, Glickman JN, Kutok JL, Aster JC, Zon LI
- Deficiency of glutaredoxin 5 reveals Fe-S clusters are required for vertebrate haem synthesis. Nature. 2005;436(7053):1035-39
Wingert RA, Galloway JL, Barut B, Foott H, Fraenkel P, Axe JL, Weber GJ, Dooley K, Davidson AJ, Schmid B, Schmidt B, Paw BH, Shaw GC, Kingsley P, Palis J, Schubert H, Chen O, Kaplan J, Zon LI
- Mutant-specific gene programs in the zebrafish. Blood. 2005;106(2):521-30
Weber GJ, Choe SE, Dooley KA, Paffett-Lugassy NN, Zhou Y, Zon LI
- Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nat Chem Biol. 2005;1(7):366-70
Stern HM, Murphey RD, Shepard JL, Amatruda JF, Straub CT, Pfaff KL, Weber G, Tallarico JA, King RW, Zon LI
- Gene expression analysis of zebrafish heart regeneration. PLoS Biol. 2006;4(8):e260
Lien CL, Schebesta M, Makino S, Weber GJ, Keating MT
- Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish. Blood. 2007;110(10):3627-36
Bahary N, Goishi K, Stuckenholz C, Weber G, Leblanc J, Schafer CA, Berman SS, Klagsbrun M, Zon LI
- Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110. Proc Natl Acad Sci USA. 2007;104(16):6608-13
Trede NS, Medenbach J, Damianov A, Hung LH, Weber GJ, Paw BH, Zhou Y, Hersey C, Zapata A, Keefe M, Barut BA, Stuart AB, Katz T, Amemiya CT, Zon LI, Bindereif A
- Prostaglandin E2 Regulates Vertebrate Hematopoietic Stem Cell Homeostasis. Nature. 2007;447(7147):1007-11
North TE, Goessling W, Walkley CR, Lengerke C, Kopani KR, Lord AM, Weber GJ, Jang IH, Grosser T, FitzGerald GA, Daley GQ, Orkin SH, Zon LI
- The Zebrafish runzel Muscular Dystrophy is Linked to the Titin Gene. Developmental Biology. 2007;309(2):180-92
Steffen LS, Guyon JR, Vogel ED, Howell MH, Zhou Y, Weber GJ, Zon LI, Kunkel LM
