Dissections and hematopoietic analyses of AGM explants. (A) Schematic cross-section through the trunk region of an E10 embryo. The boxes indicate the tissues that were dissected: AGM, aorta-gonads-mesonephros; AGM:gut, AGM with midgut, including the part of the vitelline artery within the loop of the midgut; AGM:NT, AGM with neural tube, notochord and somite remnants just lateral to the neural tube. Tissues were processed directly or after a 3-day explant culture and treated with collagenase, single cell-suspensions were prepared and cells were analyzed by FACS or injected into irradiated recipients for the in vivo transplantation assays for progenitors (CFU-S₁₁) or HSCs. (B) Number of CFU-S₁₁ after explant culture of early and late E10 AGM and AGM:gut tissues. The difference in CFU-S₁₁ numbers obtained from early E10 AGM:gut as compared with AGM after explant was statistically significant (Student's t-test, P<0.05). (C) Number of CFU-S₁₁ after explant culture of late E10 AGM and AGM:NT tissues. One to two embryo tissue equivalents (ee) of cells (wild-type or Ln72 transgenic) were injected per recipient. Results are based on seven experiments (early E10 AGM and AGM:gut), four experiments (late E10 AGM and AGM:gut) and six experiments (late E10 AGM and AGM:NT). The total number of mice injected with cells in B and C was 138, with a total of 506 CFU-S detected (average: 3.6 CFU-S/spleen). Twenty-eight irradiated (noninjected) control mice were used in these studies (one to three mice/experiment) with only two colonies detected (average: 0.06 CFU-S/spleen). (D) Number of CFU-S₁₁ per ee after explant culture of late E10 AGM, gut and AGM:gut. After explant culture some of the separately cultured AGMs and guts were pooled (ratio 1:1; referred to as `AGM+gut_mix'). Each recipient received 2 ee of cells. Total number of recipients is 8 (AGM:gut), 12 (AGM), 12 (gut) and 12 (AGM+gut_mix). No colonies were detected in irradiated (noninjected) control mice. (E) Number of CD45 Ly6a-GFP double-positive cells per ee after explant culture of early and late E10 AGM, gut and AGM:gut as determined by flow cytometric analysis. The data are based on three (early E10) and four (late E10) experiments. s.e. is indicated. Differences in CFU-S/ee in C and D and in CD45⁺ Ly6a-GFP⁺ cells/tissue in E are statistically significant (P<0.05).

HSC analysis after explant culture of AGM and AGM with surrounding tissues. Percentage of adult recipient mice repopulated with HSCs from explants of early E10 (A) and late E10 (B) AGM, gut, AGM:gut and AGM:NT tissues (from Ln72, Ly6a-lacZ or Ly6a-GFP transgenic embryos). Cells were injected into irradiated mice (1.2-1.8 ee per recipient for late E10 and 2-2.5 ee per recipient for early E10). Each column represents the number of mice repopulated per number of recipients transplanted (late E10: 21/39 for AGM, 26/42 for AGM:gut, 6/15 AGM:NT; early E10: 0/17 for AGM, 4/14 for AGM:gut, 0/5 for AGM:NT, 0/15 for gut). Error bars indicate s.d. for the combined results of 12 separate experiments. Percentages of donor cells in peripheral blood of the four recipients repopulated with early E10 AGM:gut are 86%, 93%, 60% and 98%. (C) Representative semi-quantitative PCR analysis for multi-lineage hematopoietic repopulation of primary (1° recipient 1 and 2) injected with cells from early E10 AGM:gut. Donor marker PCR was performed on DNA from hematopoietic cells and tissues. PB, peripheral blood; LN, lymph node; Sp, spleen; Th, thymus; BM, bone marrow cells; T, sorted spleen T lymphocytes; B, sorted spleen B lymphocytes; Er, sorted BM erythroid cells; Ly, sorted BM lymphoid cells; My, sorted BM myeloid cells. (D) PCR analysis performed on DNA of peripheral blood of secondary recipients transplanted with 2×10⁶ BM cells from the primary recipients (shown in C). Percentage donor marker contributions (Ln72) are indicated below each lane. Controls include the myogenin gene for DNA normalization and quantitation standards containing 100, 60, 30, 10, 6, 1 and 0% donor (Ln72) DNA.

Chimeric tissue reaggregates indicate the origin of hematopoietic progenitors/HSCs after AGM explant culture with ventral tissues. (A) Schematic diagram of reaggregate protocol. Genetically distinct AGMs and guts were combined in a cell suspension and reaggregates deposited on a filter for culture. After 3 days the reaggregate cell suspensions were injected into recipients. CFU-S were examined at 11 days post-transplantation, and HSC engraftment at 4 months post-transplantation. (B) Part of a spleen containing one CFU-S₁₁ from a recipient injected with cells from an AGM^GFP:gut^WT reaggregate. Within the circled area is one representative colony of GFP-expressing cells (white spotted area). (C) Representative PCR analysis of DNA from individual CFU-S₁₁ from recipients injected with AGM^WT:gut^GFP reaggregate cells (lanes 1-5) or AGM^GFP:gut^wt reaggregate cells (lanes 6-9). Each lane contains the PCR product of DNA isolated from one CFU-S₁₁. The myogenin (myo) normalization control indicates that all CFU-S yielded DNA (lanes 1-9). See Table 1 for complete results.

Expression of Hedgehogs, receptors and targets in the AGM. (A) RT-PCR analysis of freshly dissected AGM, AGM:NT and AGM:gut tissues, and sorted HSCs/progenitor cells (c-Kit⁺ CD34⁺), endothelial cells (c-Kit^- CD34⁺) and mesenchymal cells (c-Kit^- CD34^-) for the expression of Hedgehogs, receptors (Ptch1, Ptch2), signal transducer (Smo) and intracellular transcriptional effectors (Gli1, Gli2, Gli3). c-Kit⁺ CD34⁺, c-Kit^- CD34⁺ and c-Kit^- CD34^- represented 0.8%, 2.0% and 95.4%, respectively, of the total AGM population. Sorted cells were 83-96% pure. + indicates reaction with reverse transcriptase and - indicates no reverse transcriptase. (B) β-Galactosidase-stained (blue) transverse section through the trunk (AGM) region of an early E10 Gli1-CreERT2;R26R-lacZ embryo after overnight culture in tamoxifen. Arrows indicate Gli1⁺ mesenchymal cells and arrowhead indicates mesenchymal cells directly underlying aortic endothelial cells. Top is dorsal, bottom is ventral. (C) Gli1-expressing mesenchymal cells (blue) underlying the aortic endothelium (underlying a hematopoietic cluster) of an early E10 Gli1-CreERT2;R26R-lacZ embryo after overnight culture in tamoxifen. (D) Section through an early E10 AGM Gli1-CreERT2;R26R-lacZ explant cultured for 3 days in tamoxifen. Gli1⁺ mesenchymal cells are dispersed throughout the tissue (arrow) and underly the endothelial cells of the vessel (arrowhead). (E) Positional information influences AGM HSC induction. Hedgehog protein production is dorsal and ventral to the aorta (arrows). The Hedgehog pathway exhibits no dorsal/ventral polarity in activation, as Gli-expressing cells (blue) are found surrounding the aorta. However, ventral tissue induces the production of HSCs through gut-expressed Hedgehog protein and perhaps other ventral factors (such as Bmp4), as shown by the green gradient. Dorsal tissue inhibits AGM HSC induction as shown by the red gradient, suggesting that unknown dorsal factors (or antagonists) act to inhibit HSC induction and effect a ventral polarized induction of HSCs.