spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online August 4, 2003
doi: 10.1242/10.1242/dev.00632

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ferkowicz, M. J.
Right arrow Articles by Yoder, M. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ferkowicz, M. J.
Right arrow Articles by Yoder, M. C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

Michael J. Ferkowicz1,3, Mark Starr1,3, Xiaodong Xie1,3, Weiming Li1,3, Scott A. Johnson1,3, William C. Shelley1,3, Paul R. Morrison1,2,3 and Mervin C. Yoder1,2,3,*

1 Departments of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA
2 Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
3 Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202, USA



View larger version (62K):

[in a new window]
  		Fig. 1. Cell surface expression of CD41 (x axis) and CD34 (y axis) in yolk sac, para-aortic splanchopleure (P-Sp), aorta-gonad-mesonephros (AGM), fetal liver and adult bone marrow. (A) Isotype antibody staining of E7.0 whole embryo tissue, (B) E7.0 whole embryo tissue, (C) E8.0 yolk sac cells, (D) E9.5 yolk sac cells, (E) E9.5 P-Sp cells, (F) E10.0 yolk sac cells, (G) E10.0 AGM cells, (H) E12.5 fetal liver cells, and (I) adult marrow cells. Numbers listed in upper right corner are the percentage of total cells coexpressing CD41 and CD34 in each tissue. Representative gates used to identify CD41lo/-, CD41eim, and CD41bright cells are depicted in panel D. Results are representative data of 4 experiments.

 


View larger version (70K):

[in a new window]
  		Fig. 2. Colony morphology and analysis of hemoglobin mRNA expression. Photomicrograph of an E8.5 yolk sac primitive erythroid cells (EryP) and a definitive erythroid progenitor cell (BFU-E). Magnification x100. Panels to the right of the photomicrographs depict the results of RT-PCR analysis of plucked erythroid colonies and reveal that EryP contain mRNA for ßH1 (embryonic hemoglobin) and ß globin major (adult hemoglobin) while BFU-E express mRNA for only ß globin major. Negative control samples were tested using no reverse transcriptase.

 


View larger version (118K):

[in a new window]
  		Fig. 3. Expression of Flk-1 and CD41 in the gastrulating murine embryo. (A) E7.0 (mid-streak) embryo reveals Flk-1-expressing putative mesoderm cells migrating from the level of the embryo proper to the more proximal region of the egg cylinder (white box indicates region depicted in B and C). (B) Flk-1 staining only. (C) Emergence of CD41dim expression in the E7.0 yolk sac (arrowheads). (D) E7.25 (late-streak stage) embryo expresses both Flk-1 (E) and CD41dim (F) cells with the CD41dim cells forming a band of cells in the most proximal portion of the yolk sac while Flk-1+ cells extend toward the distal leading edge of the yolk sac as it approaches the embryo proper. (G) A composite higher magnification image of the proximal yolk sac (in orthogonal view) of an E7.25 embryo. The unlabeled visceral endoderm (endo) overlies Flk-1+ (H) and CD41dim (I) expressing cells. The arrowheads (G-I) reflect cells in transition from Flk-1+ CD41lo/- (green) to Flk-1+ CD41dim (cyan) to Flk-1dim CD41dim (blue). (J) E7.5 (neural plate stage) embryos reveal a wider distribution of Flk-1-expressing cells with appearance of numerous capillary-like structures. The band of CD41dim-expressing cells has expanded but remains centered at the proximal end of the yolk sac. Arrows indicate contaminating maternal red blood cells expressing high levels of the erythroid marker, TER119 (small bright red cells also present in D and Fig. 4B). (K) Reconfiguration of the neural plate stage embryo into a planar fashion by dividing the embryo from the most proximal to distal, results in a clear view of the continuity of the CD41dim-expressing cells for the full circumference of the yolk sac. (L) Flk-1 expression alone in the proximal region of the yolk sac. Some of the cells are Flk-1bright and many are Flk-1dim and these cells correspond to the same cells (M) that are CD41dim; the band of CD41-expressing cells is 5-10 cells in width and 1 cell thick. Flk-1 highly expressing cells overlie the band of Flk-1dimCD41dim cells but an orthogonal view (N) reveals that the Flk-1bright cells (asterisks) are interposed between the band of CD41dim cells and the outer endoderm layer (endo). Scale bar: 100 µm (A-F,J-M) and 33 µm (G-I,N).

 


View larger version (115K):

[in a new window]
  		Fig. 4. Emergence of TER119-expressing erythroid cells and CD41bright cells in the E8.0-8.25 embryo. (A) A portion of the emerging blood island in an E8.0 embryo (1-somite-pair stage) with the first TER119-expressing cells. (B) Extent of TER119 expression in cells (note the presence of TER119bright maternal erythrocytes). (C) Emergence of a new population of CD41bright cells among the CD41dim cells. (D) E8.25 (4 somite pairs) embryo that has been rendered planar with the anterior (ant) and posterior (post) regions, allantoic stalk (all), and somites (s) identified. Massive expansion in the TER119-expressing cells and extensive vasculogenesis with a network of capillary-like structures is evident as is the emergence of CD41bright cell clusters (asterisks). White box indicates region depicted in E-H. (E) High magnification composite image of a blood island containing TER119- (red), Flk-1- (green), and CD41- (blue) expressing cells. Isolated channels are shown in F-H. A red arrow in E-H identifies a cell expressing TER119 (red) but not CD41 (blue). A blue arrow (EH) indicates a cell expressing only CD41 (blue). A yellow arrow indicates a cell that is expressing TER119 (red) and CD41 (blue). Of interest the CD41bright cell in panel G (asterisk) also expresses Flk-1 (E,H). Scale bar: 100 µm (A-D) 33 µm (E-H). (I) A representative dot plot of sorted E8.25 whole embryos with quadrants color-coded to match arrows in E-H.

 


View larger version (38K):

[in a new window]
  		Fig. 5. Cell surface expression of CD41 on cells co-expressing CD34 and Kit. CD34 (y axis) and Kit (x axis) staining of (A) E9.5 yolk sac, (B) E12.5 fetal liver and (C) bone marrow cells revealed co-expression of these antigens (number in upper right corner of A-C indicates percentage of total events). (D-F) Analysis of the cells co-expressing CD34 and Kit revealed CD41 expression in (D) most E9.5 yolk sac cells, (E) lower CD41 expression in E12.5 fetal liver cells, and (F) limited CD41 expression on adult marrow cells. These are representative data from 5 experiments.

 


View larger version (7K):

[in a new window]
  		Fig. 6. Percentage donor chimerism in the blood of recipient mice transplanted with fetal liver or adult marrow cells. (A) Fetal liver donor cells expressing Kit, CD34 and CD41 (+++) demonstrated minimal but detectable repopulating ability in the competitive repopulating assay, however, cells expressing Kit, CD34, but not CD41 (++-) demonstrated high repopulating ability. (B) Adult marrow cells expressing Kit, CD34, and CD41 (+++) or minus CD41 (++-) demonstrated minimal but detectable repopulating ability in the competitive repopulation assay. Marrow cells with the phenotype Kit+CD34-CD41+ (+-+) demonstrated slightly greater repopulating ability than the +++, or ++- cells (P>0.05). Marrow cells with the phenotype Kit+CD34-CD41- (+-) demonstrated significantly greater (P<0.05) repopulating ability than all other sorted donor cell populations.

 


View larger version (46K):

[in a new window]
  		Fig. 7. Analysis of donor-derived circulating peripheral blood cells of recipient mice 6 months post-transplantation. Data from single representative recipients of fetal liver Kit+CD34+CD41- cells (A-C) or Kit+CD34-CD41- bone marrow cells (D-F) are depicted. Donor type CD45.1 expression is indicated on the y axis and the B lymphocyte marker B220 (A,D), granulocyte marker Gr-1 (B,E), and T lymphocyte markers CD4 and CD8 (C,F) are depicted on the x axis.

 


View larger version (42K):

[in a new window]
  		Fig. 8. Cell surface co-localization of CD41, CD61, and fibrinogen on E9.5 yolk sac cells. (A) CD41 (y axis) and CD61 (x axis) expression on E9.5 yolk sac cells indicates a significant percentage of the total cells co-express these markers. (B) E9.5 yolk sac cells expressing CD61 (12.1% of total cells) were isolated from non-expressing cells and further analyzed (C,D) for co-localization of CD41 and fibrinogen. (C) CD61- cells failed to demonstrate significant co-localization of CD41 and fibrinogen while CD61+ yolk sac cells (D) highly co-localized CD41 and fibrinogen (30.1% of the CD61+ cells). Data are representative of 4 experiments.

 


View larger version (21K):

[in a new window]
  		Fig. 9. Model of CD41 expression on various hematopoietic cell subsets. The results of the present studies lead us to predict a model whereby the primitive and definitive erythroid progenitor cells can be discriminated by the level of CD41 expression. Likewise, CD41 expression varies on long-term repopulating bone marrow stem cell subsets with the cells possessing the greatest repopulating ability expressing neither CD34 or CD41 (yellow cell) on the cell surface.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003