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Targeted gene disruption experiments in the mouse have demonstrated an absolute requirement for several transcription factors for the development of hematopoietic progenitors during embryogenesis. Disruption of the basic helix-loop-helix gene SCL (stem cell leukemia) causes a block early in the hematopoietic program with defects in all hematopoietic lineages. To understand how SCL participates in the organogenesis of blood, we have isolated cDNAs encoding Xenopus SCL and characterized the function of SCL during embryogenesis. We demonstrate that SCL is expressed in ventralmesoderm early in embryogenesis. SCL expression is induced by BMP-4, and a dominant negative BMP-4 receptor inhibits SCL expression in the ventral region of the embryo. Expression of SCL in either bFGF-treated animal pole explants or dorsal marginal zone explants leads to the expression of globin protein. Furthermore, over-expression of SCL does not alter normal dorsal-ventral patterning in the embryo, indicating that SCL acts to specify mesoderm to a hematopoietic fate after inductive and patterning events have occurred. We propose that SCL is both necessary and sufficient to specify hematopoietic mesoderm, and that it has a similar role in specifying hematopoietic cell fate as MyoD has in specifying muscle cell fate.
Fig. 3. Whole embryo in situ analysis of SCL expression. (a) Ventral view. SCL is first detected at stage 15 in the ventral region of the embryo. (b) Cross section of a stage 16 embryo indicates that SCL staining is present in ventralmesoderm and ectoderm. (c and d) Ventral and lateral view, respectively, of a stage 20 embryo. SCL staining increases as development proceeds. (e-g) Embryos cleared with benzyl alcohol/benzyl benzoate (1:2 v/v) to reveal staining within the embryo. (e) Stage 22. (f and g) Stage 24. (f) Staining is evident in individual spinal neurons. (g) SCL staining demarcates the ventralblood island (VBI) and extends rostrally in two stripes that cross at the mid-line. These stripes may represent vascular endothelial precursors. Staining is absent from the region of the developing liver which demarcates the anterior boundary of the ventralblood island. (h,i) Lateral and ventral-lateral views, respectively, of a stage 25 embryo. SCL staining extends caudally as the embryo grows and the VBI increases in size. (j) Stage 27; SCL staining is evident in the AGM (aorta-gonads-mesonephros) region (white arrow) and most likely indicates the development of a population of definitive hematopoietic stem cells in this region. SCL staining in neural tissue of the brain is evident at this stage (black arrow). (k) At stage 32, SCL staining in the VBI is very intense. Although neural staining (particularly at the midbrain-hindbrain boundary; arrow) is still evident, staining in the AGM region has diminished. (l) Stage 37/38; as circulation begins, blood cells leave the VBI and enter circulation and SCL staining in this region decreases. Note that the most posterior cells in the VBI are still present (white arrow). (m) Stage 40; all SCL positive cells in the VBI have entered circulation. (n,o) Double staining for SCL (purple) and GATA-2 (turquoise). (n) Double stained embryo at stage 24; SCL expression is limited to a subset of GATA-2-expressing cells on the ventral axis of the embryo. (o) Stage 25; SCL expression persists but GATA-2 expression decreases as the cells within the VBI differentiate (see text for details).
Fig. 4. SCL expression responds to BMP-4 signaling. (a) BMP-4 induces SCL expression in primitive ectoderm. Embryos were injected with BMP-4 mRNA (1 ng per embryo in the animal pole region). Animal pole explants were removed at stage 8 and treated with soluble growth factors (activin at 100 pM (Genentech); human basic FGF at 50 ng/ml; Gibco BRL) and cultured to mid-neurula stage (stage 19). Sibling whole embryo was used as a positive control. Whole embryo −RT (without reverse transcriptase) was used as a negative control. SCL and EF-1α sequences were amplified from first strand cDNA, separated on polyacrylamide gels and visualized by autoradiography. (b) Ectopic expression for BMP-4 or Mix.1 induces SCL expression in the whole embryo. Embryos were injected with either BMP-4 or Mix.1 mRNA (500 pg per blastomere at the two cell stage) and cultured to sibling tailbud stage. SCL expression was assayed by whole-mount in situ hybridization. Expression of either BMP-4 or Mix.1 caused ventralization and increased expression of SCL.
Fig. 5. Ventral expression of SCL is blocked by a dominant negative BMP receptor but not by a dominant negative FGF receptor. (a,b) Expression of a dominant negative BMP receptor blocks SCL expression on the ventral axis. Embryos were injected at the two cell stage with 1 ng dnTFRII. Injected and uninjected sibling embryos were fixed at stage 32 and stained for SCL expression by in situ hybridization. SCL expression on the ventral axis (arrows) is ablated by injection of dominant negative BMP receptor mRNA.
(c) Expression of a dominant negative FGF receptor does not block ventral expression of SCL. Embryos were injected with mRNA encoding dominant negative FGF receptor (XFD; upper embryo; n=73) or a truncated mutant of XFD lacking part of the ligand binding domain (D50; lower embryo; n=51) (2.5 ng per blastomere at the two cell stage; Amaya et al., 1991). Embryos were fixed at the tailbud stage and analyzed for SCL expression by in situ hybridization. Although trunk and tail abnormalities exist, the SCL expression on the ventral axis (arrows) was not affected by over- expression of the dominant negative FGF receptor.
Fig. 7. Over-expression of SCL in dorsal mesoderm explants leads to ectopic globin production. Both blastomeres of embryos at the two cell stage were injected with 500 pg SCL, and cultured to stage 10.25 when marginal zone explants were prepared. Whole-mount immunohistochemistry for tadpole α-globin protein was performed when embryos and explants had reached stage 35/36. (a) Sibling, uninjected ventral marginal zone (VMZ; 18/18 explants were globin positive) and whole embryo. White arrows point to globin-staining cells in the VMZ and VBI of the tadpole. (b) Uninjected dorsal marginal zone (DMZ; 0% globin positive, n=18). Note the presence of dorsal anterior structures such as eye and cement gland (black arrow). (c) SCL-loaded DMZ explants had abundant blood (white arrows; 21/22 DMZ explants were globin positive). Dorsal anterior structures such as eye and cement gland (black arrow) persist.
