Fig. 5. BMP requirements within embryonic and adult blood compartments. Embryos were injected at the four cell stage into the VMZ (A,B) or into the DMZ (C-G) close to the midline cleavage plane with 400-500 pg tBR RNA (per blastomere) to inhibit BMP signalling plus 200 pg ß-gal RNA as a lineage trace. Embryos cultured to stage 28 were fixed briefly in MEMFA then put through a ß-gal reaction and probed by whole-mount in situ hybridisation for T4-globin (A-C). Arrowheads and arrow indicate posterior VBI. Embryos grown to stage 18 were probed for SCL (D,E) or Xfli1 (F,G). (D,F) Uninjected embryos. Whereas embryos injected into the VMZ with ß-gal RNA alone or embryos injected in the DMZ with ß-gal and tBR RNA appeared normal in phenotype (A,C), embryos injected into the VMZ with ß-gal and tBR RNA produced a second axis (B). (H-K) tBR RNA (400-500 pg per blastomere) plus ß-gal RNA (200 pg per blastomere) was directed to the region of the DLP by injecting embryos in the VLMZ at the four cell stage, aiming the needle well away from the midline cleavage plane. Embryos were grown to stage 28, fixed briefly, stained for ß-gal and probed for SCL (H), Xfli1 (I) and Xlim1 (J,K). Embryos injected in this way had a second axis, lacked posterior structures and usually had ß-gal in both axes. Black arrows indicate staining in the anterior VBI for SCL (H) and Xfli1 (I). Arrowheads in (H-J) indicate where DLP signals would have been in the absence of tBR RNA. Xlim1 is absent on one side of the embryo where ß-gal (and thus tBR RNA) is located (J) but is seen on the other side of the same embryo (arrowheads), where the lineage trace is found outside the DLP region (K).