Zhang C and Evans T (1996), BMP-like signals are required after the midblas...

XB-ART-18825

Dev Genet 1996 Jan 01;183:267-78. doi: 10.1002/(SICI)1520-6408(1996)18:3<267::AID-DVG7>3.0.CO;2-7.

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BMP-like signals are required after the midblastula transition for blood cell development.

Zhang C , Evans T .

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We have investigated the process by which the primitive erythroid cells develop during early vertebrate embryogenesis. Cultured Xenopus animal cap (AC) cells transiently activate the transcription of blood cell regulatory genes GATA-1 and GATA-2 but fail to commit stably to the blood lineage. By contrast, cells of the presumptive ventral marginal zone (VMZ), are committed by the midblastula transition (MBT) to express fully on erythroid program. Growth factor BMP-4, a member of the TGF-beta family of signaling molecules, has been implicated in the process of ventral mesoderm patterning. We show that expression of BMP-4 after MBT is sufficient to induce the blood program fully in AC cells. This includes high level expression of the blood markers SCL and globin, which are not activated in AC cells from uninjected embryos. Likewise, expression of a dominant negative receptor after MBT results in relatively normal embryos, which, however, completely lack differentiated blood cells. Our results are consistent with a role for BMP or BMP-like signaling during gastrulation in the differentiation of embryonic blood.

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Species referenced: Xenopus
Genes referenced: abr bmp4 gata1 gata2 tal1 tgfb1 tsc1

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	Fig. 1. The erythroid transcriptional program is partially and transiently activated in cultured AC cells, while VMZ are competent to express the full program. a: Specific primers were used to measure quantitatively by RTfPCR the abundance of RNA from erythroid, control, or muscle genes. AC cells were explanted at stage 8 and cultured for various times prior to RNA isolation. Samples were processed in parallel from whole embryos cultured for identical periods. Culture times and corresponding embryonic stages were (1) 0 hr, stage 8; (2) 5 hr, stage 10 + ; (3) 10 hr, stage 12; (4) 15 hr, stage 14; (5) 20 hr, stage 16; (6) 25 hr, stage 20; (7) 30 hr, stage 22; (8) 42 hr, stage 30. Lam M, contains molecular size standards; the adjacent unmarked lane is an RT/PCR control in which the RT enzyme was omitted from the reaction. b: VMZ cells were isolated at stage 10 + and cultured for 15 hr (lane 1, st. 14), 25 hr (lam2, st. 201, or 40 hr (lane 3, st. 28). Following RNA isolation, transcript levels were analyzed by RT/PCR as in (a). Lane C is a control in which the RT enzyme was omitted from the reaction.
	Fig. 2. Expression of BMP-4 is sufficient to activate fully the erythroid program in AC explants. RNA was isolated from whole embryos or from cultured AC cells derived from fertilized eggs injected with 1 ng BMP-4 RNA. AC cells were explanted at stage 8. Embryos or explants were cultured until stage (1) 14, (2) 20, (3) 30, or (4) 33. LUTE C, RTiPCR control in which the RT enzyme was omitted. By stage 30 (lane 3) or 33 (lane 4), RNA for the erythroid genes is no longer detected in AC cells from uninjected embryos (not shown in this fig ure, but see Fig. 3 for these controls).
	Fig. 3. Expression of BMP-4 after MBT is sufficient to activate fully the erythroid program in AC explants. Fertilized eggs were injected with 250 pg of the CSKABMP-4 expression plasmid, which does not transcribe the BMP-4 cDNA until MBT. AC cells were isolated at stage 8 from uninjected (left) or injected (right) embryos and cultured for various times, as in Fig. 2. In this experiment, AC cells from the injected embryos are not exposed to exogenous BMP-4 until after mesoderm induction has occurred. Note that the weak MyoD signal detected in one lane was not reproducible. The comparable whole embryo controls are not shown for this figure, but see Fig. 2.
	Fig. 4. Induction of the blood program is inhibited by expression of a dominant negative BMP receptor. RNA from whole embryos (left), embryos injected with 1 ng of the ABR RNA (middle), or AC explants isolated from embryos injected with 1 ng of the ABR RNA (right) was analyzed by RTRCR for transcripts from the various genes. Total RNA was harvested when embryos reached stage (1) 14, (2) 19, (3) 28, or (4) 34. AC cells were isolated at stage 8 and cultured for (5) 16 hr, corresponding to lane 1; (6) 24 hr, corresponding to lane 2; (7) 40 hr, corresponding to lane 3. The 11 EF-la samples correspond to the same lanes, left to right. In this experiment, the controls in which AC cells from uninjected embryos were cultured alone are not shown, but the results were identical to those shown in Fig. 3.
	Fig. 6. BMP signaling is required after MBT for blood cell development. Embryos were injected at the two cell stage in each blastomere with 250 pg CSKAIABR expression plasmid or vector alone. a: Representative embryos were cultured to stage 33 and then stained with benzidine to indicate the presence of differentiating erythroid cells (blue). The extent of the developing ventral blood islands (VBI) is illustrated by the arrows on the bottom embryo, which was injected with the CSKA vector. The top embryo was injected with the CSKA/ ABR expression plasmid and does not stain with benzidine. The injected embryos appear otherwise entirely normal, except for occasional random malformations due to injection artifacts. Mock injections using dH,O or irrelevant constructs showed no abnormal phenotypes. b: Red blood cells (examples indicated by arrows) are visualized in the CSKA injected embryo (left) but are absent in the ventral vein (not visible in the photography but marked by the arrowheads) of a representative CSKAIABR injected embryo (right). Shown are living embryos cultured one day longer than those shown in a. c: Examples illustrating the range of abnormal dorsal phenotypes found in 20% of the embryos injected with a total of 500-pg expression plasmid. All these embryos [cultured as in bl are bloodless, assayed either by visualizing circulating blood cells, or by benzidine staining. The embryo marked 1 (representative of 80% of the surviving embryos) appears otherwise entirely normal, while the embryo marked 6 illustrates the most severe dorsoanterior abnormality detected. Similar anterior abnormalities were not seen in mock-injected embryos or in embryos injected with control constructs.
	Fig. 6. Blocking BMP signaling after MBT inhibits the transcription of erythroid genes in whole embryos or VMZ explants. Embryos (WE) were either uninjected (-) or injected ( +) with the CSKNABR expression plasmid (500 pg total). At stage 10 + , VMZ explants (VMZ) were isolated from some embryos. Embryos and VMZ were cultured for an additional 40 hr (stage 28) before RNA was isolated and analyzed by RT/PCR. The ability of VMZ to activate the erythroid program is largely abolished by ABR expression.