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Whether embryonic and adult blood derive from a single (yolk sac) or dual (yolk sac plus intraembryonic) origin is controversial. Here, we show, in Xenopus, that the yolk sac (VBI) and intraembryonic (DLP) blood compartments derive from distinct blastomeres in the 32-cell embryo. The first adult hematopoietic stem cells (HSCs) are thought to form in association with the floor of the dorsal aorta, and we have detected such aortic clusters in Xenopus using hematopoietic markers. Lineage tracing shows that the aortic clusters derive from the blastomere that gives rise to the DLP. These observations indicate that the first adult HSCs arise independently of the embryonic lineage.
Figure 1. Ventral (A) and Dorsal (B) Marginal Zone Contributions to Primitive and Definitive Blood Compartments (A) Time course from stage 17 to stage 35 showing ﰇ-gal localization in VMZ injected embryos. Numbers at the top right corner in- dicate stage of development. 17a, dorsal view. 17b, 22b, and 26a, ventral views. 22a, 28, and 35, lateral views. Anterior is to the left in all cases. Black arrows indicate the position of the DLP.
(B) Time course from stage 17 to stage 35 showing ﰇ-gal localization in DMZ injected embryos. Numbers at the top right corner in- dicate stage of development. 26a shows a benzyl benzoate/benzyl alcohol cleared em- bryo. Notice total absence of ﰇ-gal staining in the DLP region (black arrow). 26c, transverse section at the level of the anteriorVBI of em- bryo in 26b. 26d, transverse section of a stage 26 embryo showing GATA-2 expression in the region of the anteriorVBI. 17a, dorsal view. 17b, 22b, and 26b, top embryo, ventral views. 22a, 26a, 26b bottom embryo, and 35, lateral views. Ventral is to the bottom in all cases. In whole mounts anterior is to the left. Black arrow indicates the position of the DLP. Red arrows indicate B-gal localization in the ante- rior VBI.
Figure 2. Blastomeres Contributing to Primitive and Definitive Blood Compartments in the Xenopus Embryo Individual blastomeres of the 32-cell stage embryo were injected with beta-gal mRNA, embryos collected at stage 26 of development, and beta-gal localization detected followed by in situ hybridization where appropriate. Nomenclature of blastomeres is according to Nakamura and Kishiyama (1971). (A) Typical C4 beta-gal injected embryo. (B) Sagittal section of embryo in (A) after whole mount in situ hybridization for beta-T4 globin. Globin expression, red arrow; black arrowhead, proctodeum. S, somites. (C) Close-up of section in (B) showing that C4 does not contribute to the posteriorVBI. (D) Typical D4 beta-gal injected embryo. and indicate the A/P position of the sections depicted in (F) and (G), respectively. (E) Photoshop montage of a 10 um sagittal section of a D4 beta-gal embryo showing D4 contribution to the posteriorVBI (red arrow). Black arrowhead, proctodeum. (F) Ten micrometer transverse section at the level of the posteriorVBI of a D4 beta-gal embryo showing that D4 contributes to the posteriorVBI, the posterior-lateral mesodermal layer, and to posteriorendoderm, but does not contribute to the DLP (black arrows). n, notochord. (G) Close-up of an 80 um vibratome section of another D4 embryo at a similar A/P position to (F), showing D4 contribution to the posteriorVBImesoderm and to a few ventral endodermal cells. This section shows bilateral staining for beta-gal as a result of leakage of RNA in to the sister D4 blastomere, a phenomenon which occurred more frequently with vegetal blastomeres compared with others. (H) Typical C1 beta-gal injected embryo. The red arrow indicates beta-gal cells located in the anteriorVBI. Black arrowhead, proctodeum. Note that C1 does not contribute to the DLP. (I) Typical D1 injected embryo showing beta-gal activity in the VBI (red arrow) and in lateralendoderm, but no contribution to the DLP. Black arrowhead, proctodeum. (J) Typical D3 beta-gal injected embryo. (K) D3 injected embryo after in situ hybridization with an SCL probe. Red arrow, SCL expression in the DLP; black arrow, SCL staining in the VBI. and indicate level at which sections represented in (L) and (M) were taken.
Figure 3. In Situ Hybridization Revealing Ex- pression of XFli-1, VEGF, and SCL in the DLP- Hypochord Region (A) Time course from stage 26 to 34 showing XFli-1 expression in the DLP-hypochord re- gion. Numbers at the top right corner indicate the stage of development. 30b shows a high magnification view of the hypochord region. Position of the notochord (n) and hypochord (black asterisk) are indicated. Red arrows in- dicate the position of the migrating XFli-1 positive cells. Green arrow in 30a points to XFli-1-positive cells migrating from the dorsal fin to the hypochord. Red arrow in 34 indicates XFli-1 cells located in the forming dorsal aorta. Black arrow in 34 indicates the position of the posterior cardinal vein.
(B) Time course from stage 26 to 32 showing expression of VEGF in the hypochord. Numbers at the top right corner indicate stage of development. Position of the notochord (n) is indicated. Arrowhead indicates the hypochord. Note that VEGF expression in the hypochord decreases after stage 30, and that by stage 32, VEGF expression is no longer detected by in situ hybridization.
(C) Time course from stage 26 to stage 33 showing SCL expression in the DLP-hypochord region. Numbers at the top right corner indicate the stage of development. SCL expression in the DLP increases from stage 26 to stage 30 and then fades away with time. By stage 33, SCL expression in the DLP cannot be detected by in situ hybridization. Note that no SCL-positive cells are detected between the DLP and the midline or in the surroundings of the hypochord at any time, even at stage 30 when the strongest expression of SCL in the DLP is observed. Position of the notochord (n) and hypochord (red asterisk) are indicated. Black arrows indicate the position of the posterior cardinal veins.
All sections in transverse orientation. Sections were taken at the same level of the A axis. Dorsal is at the top in all cases.
Figure 4. Localization of Hematopoietic Precursors in the Region of the Dorsal Aorta and Their Origins (A) In situ hybridization on 10 um sections reveals expression of GATA-2 (a and b), SCL (c), Xaml (d), and GATA-3 (e) in the region of the dorsal aorta of stage 43 embryos. Notice that both GATA-2 and Xaml are expressed in clusters within the dorsal aorta, in the ventral wall of the dorsal aorta, and in mesenchymal cells below the dorsal aorta. SCL is mainly expressed in the ventral wall of the dorsal aorta and in clusters within the aorta. GATA-3 is expressed in the pronephric ducts only. GATA-2, SCL, and Xaml expression are not detected in circulating blood cells at this stage of development (data not shown). All sections are transverse. Red arrows indicate expression in the ventral wall of the dorsal aorta and in mesenchyme below the aorta. Red arrowheads indicate the endothelial lining of the dorsal aorta. Black arrowheads indicate the pronephric ducts. n, notochord; sc, spinal cord; s, somites. (B) Detection of beta-galactosidase activity at stage 43 in C3 beta-gal injected embryos. (a) location of beta-galactosidase in a stage 43 embryo which was injected with beta-gal RNA in the C3 blastomere at the 32-cell stage. Note that beta-gal is not detected in the heart, gills, and tail vessels (TV) where circulating blood cells are easily detected, indicating that C3 does not contribute to the primitive blood. (b) Ten micrometer transverse section of another C3 embryo at the A/P level of the single median dorsal aorta (just posterior to the glomus). (c) Same section magnified. Red arrowheads indicate the endothelial lining of the dorsal aorta. Red arrows indicate beta-gal positive mesenchymal cells located just ventral to the dorsal aorta. Black arrowheads indicate pronephric ducts. beta-gal positive cells are also located further anterior in the paired dorsal aortae, pronephros, and the glomus (data not shown). n, notochord; s, somites; sc, spinal cord; PD, pronephric ducts; PN pronephros; TV, tail vessels.
