The Point Mutation Of Zebrafish C-cbl Gene Ring Finger Domain Induced Myeloproliferative Disease And Its Functional Research

As the critical ability of hematopoietic stem cells, self-renewal and differenciation are tightly regulated by the transcription factors and signaling networks. Any abnormal regulation of the self-renewal and differenciation could result in hematological malignancy. Through reverse genetic approach, we have already known some genes closely related with hematopoietic stem cell self-renewal such as pten, bmi1, hoxb4, WNT, etc.. However the known genes are limited. So we performed forward genetic screen in living animals which is genome-wide saturated, phenotype driven and unbiased.Since the genetic regulational network of zebrafish hematopoietic system is highly similar with that of human, we applied the ENU chemical mutagenesis technique, and got nearly 1,000 zebrafish F2 autosomal recessive inheritance mutant families and used the whole-mount in situ hybridization to screen abnormal phenotypes in the F3 generation. We aimed to obtain the mutation influencing the development, migration and self-renewal of definitive hematopoietic stem cells. Then we performed positional cloning to get the mutated gene and assessed its role and function in the human hematopoietic stem cell disease and the malignant transformation of the leukemia stem cells.There are already some mutations related to hematopoietic genes, such as spt(tbx16) and snh(bmp7), which are associated with the early development of hematopoietic stem cells.sau(als2) and ret(band3) are associated with the downstream regulation of the hematopoietic system. However, mutations associated with stem cell are scarce.Our screening markers are runx1 and c-myb, which mark the definitive hematopoietic stem cells. So the mutations we got are closely related to HSCs. Through the three-generation forward genetic screen, we chose a mutant with highly increased expression of c-myb in kidney and caudal hematopoietic tissue(CHT).Then we analyzed the hematopoietic phenotype of the mutation, and got the following results: 1) The definitive hematopoiesis stem cells in mutant LDD731 increased dramatically, so as the myeloid cells and the erythroid cells, but not the thymus-dependent lymphocytes. The increase of HSCs can be recognized by WISH beginning at 3dpf(days post-fertilization), but can be picked out by light microscope from 5dpf through observing highly increased cells at the CHT region. 2) The primitive hematopoiesis of the mutation LDD731 is not affected. The blood vessel is normal too. The expression of neural stem cells and the germ line stem cells are also normal. So the mutation is very specific in hematopoietic system. 3) The mutation LDD731 is homozygous lethal. They die between 9-14 dpf. The heterozygote can be alive very well without any hematopoietic phenotype. 4) PH3 staining of the mutation LDD731 is positive, while TUNEL staining is negative, suggesting that the mutation affects cell proliferation. From results of the Wright-Giemsa staining, we could see more immature cells in the mutation blood smear than those in the siblings’. But the hematopoietic differentiation is not blocked. The immature cell looks bigger and possessing bigger nucleo-cytoplasmic ratio compared with the mature cell. Sudan black staining and O-dianisidine staining can also prove that the hematopoietic differentiation is not blocked and the proliferation of myeloid cell and erythroid cell are significantly increased. These symptom accord with myeloproliferative disease(MPD), also named myeloproliferative neoplasm(MPN).Then we performed positional cloning.The mutated gene located at zebrafish chromosome 15, which is c-cbl,the mutation site is at the 382 th amino acid, from histidine to tyrosine. This site is very conservative. The c-cbl gene is a proto-oncogene that encodes a RING finger E3 ubiquitin ligase. It negatively regulate the HSC self-renewal and the cell cycle. The function of its RING finger domain is to negatively regulate the receptor protein tyrosine kinase. Reports show that c-cbl mutation is closely associated with MPD or leukemia. Through our experiment, we confirmed that the proliferative phenotype may dependent on the FLT3 signaling, consisting with that in mouse and human. Through sequencing hundred MPN patients, one of them got the c-cbl mutation H398 Y, this site is just the same as the site of our mutation LDD731.From our study we could confirm that the zebrafish c-cbl H382 Y mutation results in the MPN directly.On the whole, our genetic study is an example of “genomic ping-pang”. It will help to understand the HSC self-renewal and the pathogenesis of MPN or leukemia. Mutation LDD731 is the first zebrafish MPN model in the world till now. This mutation may be a good drug screening model for MPN or leukemia. This will also be helpful to understand the complicated HSCs self-renewal network.