Supplementary Information

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A point mutation of zebrafish c-cbl gene
ring finger domain induced a phenotype
mimic human myeloproliferative disease
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Running title: Zebrafish c-cbl mutant induces HSPC proliferation
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Xiaolan Peng1,*, Mei Dong2,*, Lie Ma5,1*, Xiao-E Jia2, Jianhua
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Mao1, Changbin Jin2, Yi Chen1, Lei Gao2, Xiaohui Liu1, Ke Ma2,
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Lei Wang2, Tingting Du1, Yi Jin1, Qiuhua Huang1, Keqin Li1,
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Leonard I. Zon3,4, Tingxi Liu1,2, Min Deng2, #, Yong Zhou2, #,
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Xiaodong Xi1, Yi Zhou3, #, Saijuan Chen1,#
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1. State Key Laboratory for Medical Genomics, Shanghai
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Institute of Hematology, RuiJin Hospital, Shanghai Jiao Tong
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University (SJTU) School of Medicine, and Collaborative
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Innovation Center of Systems Biomedicine, SJTU, Shanghai,
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200025, China
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2. Key Laboratory of Stem Cell Biology, Institute of Health
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Sciences, Shanghai Institutes for Biological Sciences and
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Graduate University, Chinese Academy of Sciences & Shanghai
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Jiao Tong University School of Medicine, Shanghai, 200025,
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China
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3. Stem Cell Program at Boston Children’s Hospital,
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Hematology/Oncology Program at Children's Hospital and Dana
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Faber Cancer Institute, Harvard Medical School, Boston,
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MA02115, USA
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4. Howard Hughes Medical Institute
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5. Shanghai Center for Systems Biomedicine, Ministry of
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Education Key Laboratory of Systems Biomedicine, Shanghai
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Jiao Tong University, Shanghai, 200240, China
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* These authors contribute equally to this work.
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# Address correspondence to:
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Saijuan Chen, E-mail: sjchen@stn.sh.cn
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Yi Zhou, E-mail: yzhou@enders.tch.harvard.edu
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Yong Zhou, E-mail: zhouyong@sibs.ac.cn
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Min Deng, E-mail: mdeng@sibs.ac.cn
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The supplementary information includes seven figures, two movies and
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one table: Supplementary Figures 1–3 and 5-6 further displayed the
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phenotype of homozygous LDD731 mutants. Supplementary Figures 4
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and 7 further confirmed c-cbl mutation. Supplementary table lists the
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oligos used in the article.
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Supplementary Figure legends
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Supplementary Figure 1.
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Increase of HSPCs began from 3 dpf. WISH analysis of
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hematopoietic lineages of LDD731 mutants and their siblings at
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3 dpf. (A–D) WISH analysis of the level and pattern of c-myb
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expression at 36 hpf and 3 dpf. (E–H) WISH analysis of the
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levels and patterns of αe1 and mpo at 3 dpf.
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Supplementary Figure 2.
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Primitive hematopoiesis was not affected in homozygous
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LDD731 mutants. (A and B) Angiogenesis and vasculogenesis
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were normal as revealed by flk1, a marker of vascular
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endothelial cells. (C–L) Expression of scl (marker for HSPCs),
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gata1 (marker for erythroid progenitors), pu.1 (marker for
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myeloid progenitors) and mpo (marker for mature myeloid cells)
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was normal in LDD731 mutants at 22 hpf, a period at the stage
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of primitive hematopoiesis. Abbreviation: anterior lateral plate
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mesoderm (ALPM), inner cell mass (ICM), dorsal aorta (DA),
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posterior cardinal vein (PCV).
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Supplementary Figure 3.
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Expression of c-myb obviously increased at the KM but not
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at
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stem/progenitor cells was specific for hematopoietic system
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in homozygous LDD731 mutants. (A and B) Dorsal view of
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c-myb expression at the KM and thymus. (C–E) WISH with
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zgc55605, a marker of pronephric ducts. (D–F) WISH
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experiment with rag-1, a marker of T cells. (G and H) Lateral
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view of the trunk showing an increased expression of c-myb
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(asterisk) in LDD731 mutants. (I and J) WISH analysis of the
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HSPC marker scl at 5 dpf. (K–N) WISH analysis of stem cell
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markers of other tissues/organs, such as vasa (marker for germ
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line stem cells) and nr4a2b (marker for neural stem cells).
the
thymus,
and
the
enhanced
proliferation
of
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Supplementary Figure 4.
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Confirmation of c-cbl mutation. (A–D) Sequencing of the
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mutation site of the c-cbl gene in genomic DNA from the
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grandfather, grandmother, father and mother of the LDD731
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mutants. Note that the father and mother were heterozygotes for
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the mutation. The results together with those of the offsprings
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(see Figure 2 in the text) agree with Mendel’s law of autosomal
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recessive inheritance. (E–I) Sequencing of the mutation site of
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the c-cbl gene in genomic DNA samples from five WT zebrafish
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strains. Genomic DNA samples from five strains of WT
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zebrafish of Tu, AB, WIK, Longfin and SH were sequenced
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excluding the genetic polymorphism. (J) Expression pattern of
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c-cbl in WT zebrafish. (K–L) RT-PCR product sequencing of
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uninjected WT embryos or c-cbl-splicing MO-injected embryos.
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Supplementary Figure 5.
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Vascular morphology of c-cbl–splicing MO-injected 5 dpf
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embryos. (A–C) Uninjected embryo of transgenic zebrafish
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with
flk1
labled
by
mCherry
(flk1:
mCherry). (D–F)
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c-cbl-splicing MO-injected embryo in Tg (flk1: mCherry).
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Caudal vessels (CV), dorsal longitudinal anastomotic vessel
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(DLAV), dorsal aorta (DA), posterior cardinal vein (PCV),
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intersegmental vessels (ISV), caudal aorta (CA), and caudal vein
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(CV).
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Supplementary Figure 6.
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Increased proliferation of HSPCs from 3 dpf in LDD731.
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(A–H) Confocal image of c-myb+ and pH3+ cells in CHT
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between homozygous LDD731 mutants and their siblings at 3
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dpf. pH3+ cells were labeled with GFP and c-myb+ cells with
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RFP. (I) Statistical analysis of the numbers of pH3+ cells in
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CHT between homozygous LDD731 mutants and their siblings
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at 3 dpf. (J) Statistical analysis of pH3+ cells and c-myb+ cells
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at CHT in homozygous LDD731 mutants and their siblings at 3
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dpf (t-test, p<0.05).
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Supplementary Figure 7.
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Validation of the CBL-FLT3 pathway. (A–D) Wild-type
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embryos were treated with or without PKC412 or Lestaurtinib at
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2–5 dpf and fixed for c-myb in situ hybridization.
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Supplementary Movie 1
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Circulation of CHT region of 5 dpf in heterozygous LDD731
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siblings or wild type siblings. Caudal aorta (CA); Caudal vein
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(CV).
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Supplementary Movie 2
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Circulation of CHT region of 5 dpf in homozygous LDD731
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mutants. Caudal aorta (CA); caudal vein (CV).
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