Whole-mount Three-dimensional Imaging of Internally Localized Immunostained Cells Within Mouse Embryos

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2012 Feb 11

PMID 22322215

Citations 97

Authors

Tomomasa Yokomizo

Tomoko Yamada-Inagawa

Amanda D Yzaguirre

Michael J Chen

Nancy A Speck

Elaine Dzierzak

Affiliations

Soon will be listed here.

Abstract

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

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References

Medvinsky A, Rybtsov S, Taoudi S . Embryonic origin of the adult hematopoietic system: advances and questions. Development. 2011; 138(6):1017-31. DOI: 10.1242/dev.040998. View

Tavian M, Coulombel L, Luton D, Clemente H, Dieterlen-Lievre F, Peault B . Aorta-associated CD34+ hematopoietic cells in the early human embryo. Blood. 1996; 87(1):67-72. View

Yokomizo T, Ogawa M, Osato M, Kanno T, Yoshida H, Fujimoto T . Requirement of Runx1/AML1/PEBP2alphaB for the generation of haematopoietic cells from endothelial cells. Genes Cells. 2001; 6(1):13-23. DOI: 10.1046/j.1365-2443.2001.00393.x. View

Ghiaur G, Ferkowicz M, Milsom M, Bailey J, Witte D, Cancelas J . Rac1 is essential for intraembryonic hematopoiesis and for the initial seeding of fetal liver with definitive hematopoietic progenitor cells. Blood. 2007; 111(7):3313-21. PMC: 2275001. DOI: 10.1182/blood-2007-08-110114. View

Zovein A, Turlo K, Ponec R, Lynch M, Chen K, Hofmann J . Vascular remodeling of the vitelline artery initiates extravascular emergence of hematopoietic clusters. Blood. 2010; 116(18):3435-44. PMC: 8127023. DOI: 10.1182/blood-2010-04-279497. View

Becker K, Jahrling N, Kramer E, Schnorrer F, Dodt H . Ultramicroscopy: 3D reconstruction of large microscopical specimens. J Biophotonics. 2009; 1(1):36-42. DOI: 10.1002/jbio.200710011. View

Yokomizo T, Dzierzak E . Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development. 2010; 137(21):3651-61. PMC: 2964097. DOI: 10.1242/dev.051094. View

Ng C, Yokomizo T, Yamashita N, Cirovic B, Jin H, Wen Z . A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells. Stem Cells. 2010; 28(10):1869-81. DOI: 10.1002/stem.507. View

Ferkowicz M, Yoder M . Whole embryo imaging of hematopoietic cell emergence and migration. Methods Mol Biol. 2011; 750:143-55. DOI: 10.1007/978-1-61779-145-1_10. View

10.

Garcia-Porrero J, Godin I, Dieterlen-Lievre F . Potential intraembryonic hemogenic sites at pre-liver stages in the mouse. Anat Embryol (Berl). 1995; 192(5):425-35. DOI: 10.1007/BF00240375. View

11.

Yoshida H, Takakura N, Hirashima M, Kataoka H, Tsuchida K, Nishikawa S . Hematopoietic tissues, as a playground of receptor tyrosine kinases of the PDGF-receptor family. Dev Comp Immunol. 1998; 22(3):321-32. DOI: 10.1016/s0145-305x(98)00008-1. View

12.

Garcia-Porrero J, Manaia A, Jimeno J, Lasky L, Dieterlen-Lievre F, Godin I . Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites. Dev Comp Immunol. 1998; 22(3):303-19. DOI: 10.1016/s0145-305x(98)00006-8. View

13.

Zucker R, Hunter 3rd E, Rogers J . Confocal laser scanning microscopy of morphology and apoptosis in organogenesis-stage mouse embryos. Methods Mol Biol. 2000; 135:191-202. DOI: 10.1385/1-59259-685-1:191. View

14.

Marshall C, Thrasher A . The embryonic origins of human haematopoiesis. Br J Haematol. 2001; 112(4):838-50. DOI: 10.1046/j.1365-2141.2001.02537.x. View

15.

Gard D . Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs. Methods Cell Biol. 1993; 38:241-64. DOI: 10.1016/s0091-679x(08)61006-7. View

16.

Yokomizo T, Ng C, Osato M, Dzierzak E . Three-dimensional imaging of whole midgestation murine embryos shows an intravascular localization for all hematopoietic clusters. Blood. 2011; 117(23):6132-4. PMC: 3122938. DOI: 10.1182/blood-2011-02-334037. View

17.

Jaffredo T, Richard C, Pouget C, Teillet M, Bollerot K, Gautier R . Aortic remodelling during hemogenesis: is the chicken paradigm unique?. Int J Dev Biol. 2010; 54(6-7):1045-54. DOI: 10.1387/ijdb.103062tj. View

18.

Dzierzak E, Speck N . Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol. 2008; 9(2):129-36. PMC: 2696344. DOI: 10.1038/ni1560. View