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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 ventral mesoderm 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 ventral blood 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 ventral blood 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). |
