The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis that allows for the critical in vivo analysis of cell development and lineage determination. Conservation of the variety of blood cell types and key regulatory factors between mammals and zebrafish suggests that the regulatory mechanisms directing lineage specification are similarly conserved. To identify novel genes necessary for normal myeloid cell development, we have analyzed a panel of zebrafish insertional mutants, provided by Nancy Hopkins at Massachusetts Institute of Technology (MIT), and identified mutants with deficient or abnormal distribution of myeloperoxidase (mpo), a gene specifically expressed in zebrafish granulocytes. One of the mutants identified in this screen is disrupted in fbxo5, a gene not previously identified as having a role in hematopoiesis. Using whole mount RNA in situ analysis on mutant and morpholino-injected embryos (morphants) we have determined that decreased levels of fbxo5 result in severely diminished numbers of granulocytes, although erythropoiesis appears normal. Interestingly, fbxo5 morphants have normal levels of pu.1 and l-plastin expression. Since we have previously shown that knockdown of pu.1 results in the loss of mpo-expressing cells, our data suggest that fbxo5 may function downstream or in a parallel pathway to pu.1 during granulopoiesis. To determine if the decreased number of granulocytes was due to programmed cell death, we examined the level of apoptosis in morphant versus control embryos. Whole mount acridine orange staining of apoptotic cells indicated that fbxo5 morphants had increased apoptosis, which can be mostly rescued by co-injection with p53 morpholino. However, the fbxo5/p53 double morphants continued to display a severe decrease in granulocytes, suggesting that the loss of granulocytes is not due to p53-mediated apoptosis. Fbxo5 has been examined previously in Xenopus oocytes, where the protein was found to regulate cell cycle progression by inhibiting APC. Interestingly, we have identified multiple mutants in genes that normally regulate the G2/M checkpoint and mitosis during the cell cycle all which display decreased numbers of granulocytes, suggesting a role for the regulation of the cell cycle in granulopoiesis. While further analysis is needed to determine where and how fbxo5 functions during myeloid cell development, as well as each of the genes disrupted in our additional cell cycle mutants, we have identified that fbxo5 is necessary specifically for the normal development of granulocytes.

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