Unexpected requirement for ELMO1 in clearance of apoptotic germ cells in vivo

Journal name:
Nature
Volume:
467,
Pages:
333–337
Date published:
DOI:
doi:10.1038/nature09356
Received
Accepted

Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases1, 2, 3. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells4, 5, 6, 7. Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo.

At a glance

Figures

  1. Disrupted seminiferous tubule architecture in ELMO1-deficient mice.
    Figure 1: Disrupted seminiferous tubule architecture in ELMO1-deficient mice.

    a, Immunolocalization of ELMO1 and DOCK1 in Sertoli cells (arrows) of adult C57BL/6J mice testes. Control, secondary antibody only. b, Immunoblotting of lysates from testes of adult mice with the indicated Elmo1 genotypes. A non-specific (NS) band served as an internal loading control. WT, wild type. c, mRNA levels of Elmo1, Elmo2 and Elmo3 in Sertoli cells relative to Gapdh and normalized to wild type for each gene. *P = 0.01, n = 3 mice per group. d, Average number of giant cells per tubule in testes of six-week-old mice. *P = 0.03, n = 3 or 4 mice per group. e, H&E sections of testes of mice with the indicated Elmo1 genotypes. Magnification, ×10 (upper panels) and ×25 (lower panels). Arrow, a giant cell; arrowheads, vacuolar spaces in the epithelium. Error bars, s.e.m.

  2. Requirement for ELMO1 in apoptotic cell clearance in vivo and in vitro.
    Figure 2: Requirement for ELMO1 in apoptotic cell clearance in vivo and in vitro.

    a, Detection of apoptotic cells in the testes of wild-type and Elmo1/ mice by immunohistochemistry (apostain, brown) viewed under ×10 (left) and ×25 (right) magnification. Arrows, apoptotic cells. b, Average number of apoptotic nuclei per tubule. *P = 0.04, n = 3 or 4 mice per group. c, Total number of Sertoli cells isolated from testes of individual mice. n = 4 mice per group. Error bars in b and c indicate s.e.m. d, e, Fluorescence microscopy of wild-type and Elmo1−/− Sertoli cells. d, Staining with antibodies against the Sertoli cell marker anti-Mullerian hormone (anti-AMH, green) or control immunoglobulin-G (inset). e, Sertoli cells were fed 2.1-μm carboxylate beads (red) or apoptotic mouse spermatic germ cells labelled with CypHer5 (red). Green, phalloidin staining; blue, 4′,6-diamidino-2-phenylindole-stained nuclei. Arrows, cells with engulfed targets; arrowheads, individual engulfed targets. Scale bar, 50μm. fh, In vitro phagocytosis by wild-type and Elmo1−/− Sertoli cells. Uptake of fluorescent 2.1-μm carboxylate beads (f) and CypHer5-labelled apoptotic germ cells (h) was quantified by flow cytometry. *P<0.005, n = 4 mice per group. Error bars, s.d. g, Representative flow cytometry histograms of data in h. CytoD, 10μM cytochalasin D.

  3. ELMO1 functions in Sertoli-cell-mediated apoptotic germ cell clearance.
    Figure 3: ELMO1 functions in Sertoli-cell-mediated apoptotic germ cell clearance.

    a, Detection of apoptotic cells (apostain, brown) in testes of six- to eight-week-old Amh-Cre/Elmo1F/W and Amh-Cre/Elmo1F/F mice. Arrows, apoptotic cells. Magnification, ×12.5 (upper panels) and ×25 (lower panels). b, Quantitative PCR of Elmo1 and Elmo2 mRNA levels in Sertoli cells from Amh-Cre+ mice with the indicated genotypes. Error bars, s.d. c, Average number of apoptotic cells per tubule. *P = 0.04, n = 5 or 6 mice per group. Error bars, s.e.m. d, Total sperm output in eight-week-old Amh-Cre+ Elmo1F/F mice and littermate controls. *P = 0.007, n = 6–8 testes per group. Error bars, s.d. e, Phagocytosis by TM4 Sertoli cells expressing GFP, GFP-tagged wild type or dominant-negative ELMO1 (T625) expression constructs. *P<0.05, n = 3. Error bars, s.d. f, RAC1-GTP levels in TM4 Sertoli cells transfected with the indicated constructs. RAC1-GTP relative to actin was quantified by densitometry. g, Uptake of 2.1-μm carboxylated targets by TM4 Sertoli cells following siRNA knockdown of Rac1. *P = 0.02, n = 3. Inset, immunoblot of TM4 lysates after Rac1 knockdown. DOCK1, loading control. Error bars, s.d.

  4. BAI1 participates as an engulfment receptor in Sertoli-cell-mediated engulfment.
    Figure 4: BAI1 participates as an engulfment receptor in Sertoli-cell-mediated engulfment.

    a, Phosphatidylcholine (PC, blue) or phosphatidylserine (PtdSer, red) microbubble uptake by TM4 Sertoli cells. *P<0.05, n = 4. Error bars, s.d. b, Levels of PtdSer recognition receptors by quantitative PCR from the indicated murine tissues. #, mRNA below detectable level. One representative experiment of three is shown. BMDM, bone-marrow-derived macrophages. c, Phagocytosis by TM4 Sertoli cells transfected with control or Bai1 siRNA. *P = 0.03, n = 3. Error bars, s.d. d, Schematic of microinjection into rete testis (left), and rete testis and efferent ducts before (top right) and 24h after injection of fluorescent microspheres under light (middle right) and fluorescence (lower right) microscopy of whole mounted seminiferous tubule. Dashed lines, tubule wall borders; arrow, cluster of microspheres internalized by Sertoli cells. e, 15P-1 Sertoli cells on Matrigel incubated with fluorescent targets along with GST or BAI1-TSR protein. f, g, Intratesticular injection of GST or BAI1-TSR into six- to eight-week-old C57BL/6J mice. f, Apoptotic cells detected by immunohistochemistry (apostain, brown) after 24h. Magnification, ×12.5 (upper panels) and ×25 (lower panels). Arrows, apoptotic cells. g, Sperm counts normalized to testis weight. *P<0.05, n = 3. Error bars, s.e.m.

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Author information

  1. These authors contributed equally to this work.

    • Jeffrey J. Lysiak &
    • Kodi S. Ravichandran

Affiliations

  1. Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia 22908, USA

    • Michael R. Elliott,
    • Daeho Park,
    • Ignacio J. Juncadella &
    • Kodi S. Ravichandran
  2. Center for Cell Clearance, University of Virginia, Charlottesville, Virginia 22908, USA

    • Michael R. Elliott,
    • Daeho Park,
    • Ignacio J. Juncadella,
    • Jason M. Kinchen,
    • Jeffrey J. Lysiak &
    • Kodi S. Ravichandran
  3. Department of Urology, University of Virginia, Charlottesville, Virginia 22908, USA

    • Shuqiu Zheng,
    • Robin I. Woodson,
    • Michael A. Reardon,
    • Jun Zhang &
    • Jeffrey J. Lysiak
  4. Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA

    • Jason M. Kinchen &
    • Kodi S. Ravichandran

Contributions

The project was designed and the experiments planned by J.J.L., K.S.R and M.R.E. M.R.E. generated the Elmo1 knockout mice and performed and analysed most of the experiments in this study. S.Z. carried out the experiments using ELMO–GFP constructs. R.I.W. performed all of the immunohistochemistry, Sertoli cell extraction and sperm counts. M.A.R. performed the microbubble uptake experiments. I.J.J. performed the phagocytosis assays on non-Sertoli cells. D.P. performed phagocytosis assays and synthesized and characterized BAI1-TSR. Caenorhabditis elegans mismigration analysis using Elmo1 transgenes was conducted by J.M.K. J.Z. assisted with immunoblotting and quantitative PCR studies of Sertoli cells. J.J.L. performed intratesticular injections. M.R.E., K.S.R. and J.J.L. wrote the manuscript with input from the co-authors.

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The authors declare no competing financial interests.

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