Essential Role of Kindlin 3 in Zebrafish Thrombopoiesis.

The zinc finger transcription factor GATA-1 is required for proliferative inhibition and terminal maturation of megakaryocytes, and is mutated in Down Syndrome Transient Myeloproliferative Disorder (TMD) and Acute Megakaryoblastic Leukemia (DS-AMKL). Yet the molecular mechanisms that regulate GATA-1 activity in megakaryopoiesis remain incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. In order to further understand GATA-1’s function, and possibly identify new factors involved in megakaryopoiesis, we purified GATA-1 containing multiprotein complexes from the murine L8057 megakaryocytic cell line. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and then performed a tandem anti-FLAG immunoaffinity and streptavidin affinity purification. Using mass spectrometry (LC/MS/MS), we identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β. SP1 and all components of the NuRD complex (which binds FOG-1) as co-purifying proteins. In addition, we reproducibly obtained several novel proteins. We previously reported the identification of the kruppel-type zinc finger transcription factor zfp148 (also called ZBP-89), and showed that it plays an essential role in megakaryopoiesis and definitive erythropoiesis. Here we report the identification of Kindlin 3 (also called URP2 for UNC-112 related protein 2), a member of a family of PH and FERM domain containing proteins that are thought to play a role in integrin-mediated processes. Expression of Kindlin 3 is restricted to hematopoietic cells, principally megakaryocytes and lymphocytes. It is first expressed at ~E9.5 during murine embryogenesis, and is abundant in fetal liver megakaryocytes by day E14.5. In order to begin to assess the role of Kindlin 3 in megakaryopoiesis in vivo, we performed morpholino-mediated knockdown of Kindlin 3 expression in CD41-GFP transgenic zebrafish embryos. In contrast to control embryos, embryos injected with Kindlin 3 specific morpholinos exhibited nearly complete loss of GFP+ thrombocytes (equivalent to mammalian megakaryocyte/platelets). Erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development) was not significantly affected, similar to embryos injected with zfp148-specific morpholinos. Given the role of integrin outside-to-inside signaling in megakaryopoiesis, we propose that Kindlin 3 may play a role linking extracellular signals to megakaryocyte maturation and growth control via GATA-1 transcription complexes. Further analysis in murine systems is underway to test this hypothesis.