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Browsing by Author "Zhao, Liang"
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Item Generation and mutational analysis of a transgenic mouse model of human SRY(Wiley, 2022-03) Thomson, Ella; Zhao, Liang; Chen, Yen-Shan; Longmuss, Enya; Ng, Ee Ting; Sreenivasan, Rajini; Croft, Brittany; Song, Xin; Sinclair, Andrew; Weiss, Michael; Pelosi, Emanuele; Koopman, Peter; Biochemistry and Molecular Biology, School of MedicineSRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage the pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a human SRY transgene able to drive testis determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each of SRY's three domains (N-terminal, HMG box, and C-terminal) and performed a detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to human SRY and present a versatile and powerful system in which to functionally analyze variations of SRY including known and novel pathogenic variants found in DSD.Item Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mic(American Society of Hematology, 2018-09-06) Capitano, Maegan; Zhao, Liang; Cooper, Scott; Thorsheim, Chelsea; Suzuki, Aae; Huang, Xinxin; Dent, Alexander L.; Marks, Michael S.; Abrams, Charles S.; Broxmeyer, Hal E.; Microbiology and Immunology, School of MedicineWe hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/β-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/β-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/β-/- BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/β-/- mice had higher concentrations of TGF-β1. CM from pitpα-/- and pitpα-/-/β-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-β antibody, and treatment of pitpα-/-/β-/- mice in vivo with anti-TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/β-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/β-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1.