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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02596

Development 133, 4183-4192 (2006)
Published by The Company of Biologists 2006
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The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have hematopoietic potential

Brandon M. Zeigler1, Daisuke Sugiyama1,*, Michael Chen1, Yalin Guo1, Karen M. Downs2,{dagger} and Nancy A. Speck1,{dagger}

1 Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
2 Department of Anatomy, 1300 University Avenue, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706, USA.


Figure 1
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  		Fig. 1. Runx1 expression before and after chorio-allantoic fusion. (A) Schematic diagram of an early headfold (EHF) stage mouse embryo, showing locations of the ectoplacental cavity (epc), chorion (ch), exocoelomic cavity (ecc), amnion (am), amniotic cavity (ac), embryo (emb), yolk sac blood islands (bi) and allantois (al). Shown in color and labeled are both embryonic and extra-embryonic endoderm layer (end), mesoderm layer (mes) and ectoderm layer (ect). The conceptus is surrounded by embryonic (emb), yolk sac (ys) and ectoplacental (ep) visceral endoderm (vis end). The chorion consists of chorionic mesoderm (ch mes) and ectoderm (ch ect), the latter of which is derived from trophectoderm. The anterior (a) and posterior (p) boundaries of the primitive streak (ps) are indicated. Compass shows anterior (ant), posterior (pos), proximal (pro) and distal (dis) directions for the extra-embryonic compartment. (B) Sagittal section of an EHF-stage Runx1lz/+ conceptus showing Runx1 expression (blue) in the chorionic (ch) mesoderm (black arrow) and blood islands (bi). The blue arrow indicates area of punctate blue staining in ectoplacental visceral endoderm. ß-Galactosidase staining was performed for 18 hours. (C) Runx1 expression (arrowheads) at the chorio-allantoic fusion junction in a 12 s Runx1lz/+ conceptus. (D) Detail of Runx1 expression from boxed region in C. (E) Runx1 expression in a 12 s Runx1lz/+ conceptus at the plane of chorio-allantoic fusion (black arrow), at the junctions between the allantois and chorion (blue arrows) and in a cluster of cells within the base (boxed region). ß-Galactosidase staining was performed for 18 hours. (F) Detail of Runx1 expression from boxed region in E. (G) Runx1 expression in the distal region (arrowhead) and within the allantoic base (box) in a 16 s Runx1lz/+ conceptus. (H) Detail of Runx1 expression (boxed region in G) in putative endothelial cells (arrowheads) lining a blood vessel within the allantoic base. (I) Section through placenta of a 9.5 dpc Runx1lz/+ embryo showing Runx1+ cells in allantoic mesothelium (arrow). (J) Detail of boxed region from I. (K) Runx1 expression in endothelium and umbilical artery hematopoietic clusters in a 10.5 dpc Runx1lz/+ conceptus. (L) Whole mount showing Runx1 expression in the chorionic disk (cd) and along the length of the umbilical artery (ua) of a 10.5 dpc Runx1lz/+ conceptus.

 

Figure 2
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  		Fig. 2. Isolation of the allantois and chorion. (A) Schematic diagram illustrating isolation of the allantois (al) with a glass microcapillary pipette. Several cell layers were trimmed from the base with a glass scalpel. am, amnion; ys, yolk sac. (B) Chorion (ch) isolation. Endoderm was labeled by submerging the conceptus in ConA 594 (green) and injecting ConA 488 (red) into the exocoelomic cavity (ecc) to label the mesoderm that lines the cavity. bi, blood island; epc, ectoplacental cavity. Two incisions (dotted lines) were made to isolate the chorionic plate, which is shown in the right hand panels. We then trimmed visceral endoderm (ve, green) away from the chorion (ch, red) with a glass scalpel. After 24 hours of explant culture, chorions isolated in this way had no visible traces of green fluorescent ve-derived cells (Fig. 4A,B). (C) Isolation of anterior embryonic ectoderm (control for Table 1). The endoderm was labeled with ConA 594 (green). Three cuts were made with glass scalpels: 1, a transverse cut proximal to the amnion; 2, a sagittal cut to isolate the anterior portion of the embryo; 3, the embryonic endoderm and mesoderm were removed from the ectoderm with pancreatin/trypsin digest followed by excision with a glass scalpel.

 

Figure 3
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  		Fig. 3. Runx1 expression in the allantois and chorion does not require chorio-allantoic fusion. (A) Allantois (al) explant culture on plastic (48 hours) from a Runx1lz/+ conceptus showing Runx1 expression (blue) (3.2x). (B) Chorion (ch) explant culture on plastic from a Runx1lz/+ conceptus (3.2x). (C) Allantois sphere cultures (48 hours) from Runx1lz/+ (lz/+) and wild-type (+/+) conceptuses (left). Right hand panels are nuclear fast red stained histological sections of whole mount preparations of allantoic spheres showing Runx1 expression predominantly in mesothelium on the surface of the sphere. (D) Tg(Ly6a/GFP) (Sca-1) expression from the freshly isolated allantois and chorion and following 24 hours of culture on OP9 cells.

 

Figure 4
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  		Fig. 4. The pre-fusion allantois and chorion have hematopoietic potential. (A) Explant of ConA 488 (red) labeled chorion following 48 hours of culture on OP9 stromal cells. ConA 488 positive cells that crawled off the explant are boxed. (B) Explant of ConA 594 labeled visceral endoderm (ve) after 48 hours on OP9 cells. (C) Allantois cultured for 7 days on OP9 cells in the presence of SCF, IL3, IL6, Flt3-L, G-CSF, GM-CSF and 2-ME. Note the presence of abundant, nonadherent round cells. (D) Allantois OP9 culture from a Runx1 deficient (Runx1lz/rd) conceptus. No nonadherent round cells are present. (E) Chorion OP9 explant culture, same conditions as in panel C. (F) Chorion OP9 culture from a Runx1 deficient (Runx1lz/rd) conceptus. (G) Allantois OP9 culture from a ß-actin/GFP transgenic conceptus, showing that nonadherent cells originate from the conceptus. (H) Allantois OP9 culture from ß-actin/GFP transgenic conceptus, stained with DiI-Ac-LDL (red) and anti-CD45 (blue). GFP+ DiI-Ac-LDL+ cells are yellow and GFP+ CD45+ cells are cyan. (I) Chorion OP9 culture from a ß-actin/GFP transgenic conceptus. (J) Chorion OP9 culture from a ß-actin/GFP transgenic conceptus, labeled with DiI-Ac-LDL and anti-CD45 as in panel H. (K) Representative FACS analysis of three pooled allantois and chorion explants from +/+ and Runx1 deficient (Runx1rd/rd) conceptuses cultured on OP9 cells for 14 days in the presence of SCF, IL3, IL6, Flt3-L, G-CSF, GM-CSF and 2-ME and stained for CD45 and c-kit. The explants were cultured separately, and three allantoises or chorions were pooled for FACS analysis in these plots. Cells in each gate were analyzed in the plots below. The experiment was performed three times, with a total of ten +/+ and seven Runx1rd/rd allantoises and chorions.

 

Figure 5
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  		Fig. 5. Myeloid and definitive erythroid potential of allantois and chorion explants. (A) FACS analysis of ten pooled allantois and nine pooled chorion explants following culture for 14 days on OP9 stromal cells in the presence of SCF, IL3, IL6, Flt3-L, G-CSF, GM-CSF and 2-ME. nexperiments=4, nallantois=32, nchorion=25. (B) May-Grunwald Giemsa stained nonadherent cells isolated from allantois and chorion explants cultured on OP9 cells under the conditions described in A. a-h, allantois explants; i-p, chorion explant cultures; a-c, i-n, monocytes; d-e, macrophages; f-h, o-p, myelocytes. (C) Methylcellulose colony forming assays from the allantois and chorion. Six conceptus equivalents of allantoises and chorions were cultured together as explants, disaggregated and plated in methycellulose. The top three panels are representative colonies. (Bottom) May-Grunwald Giemsa-stained cytospin preparations of individual colonies from the allantois and chorion cultures. e, erythryocyte; m, myeloid cell; bar=10 µm. Colony numbers per six conceptus equivalents (±s.e.m. in parentheses) following 2 days of explant culture in the presence of 50% rat serum on plastic, or after 5 days of explant culture on OP9 cells in the presence of SCF, Flt3-L and EPO were averaged from three independent experiments. (D) RT-PCR for globin gene expression ({epsilon}y and ßmajor). BFU-E or CFU-GM represents individual colonies from either the allantois or chorion methylcellulose cultures. Yolk sac was isolated from ~9.0 dpc conceptuses as a positive control for {epsilon}y expression, and bone marrow was used as a negative control.

 




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