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Primitive erythropoiesis in the Xenopus embryo: the synergistic role of LMO-2, SCL and GATA-binding proteins

Paul E. Mead^1,*, Anne E. Deconinck^1,2, Tara L. Huber¹, Stuart H. Orkin^1,2 and Leonard I. Zon^1,2,

¹ Division of Hematology/Oncology, Department of Pediatrics, Harvard Medical School,
² Howard Hughes Medical Institute, Children’s Hospital, 300 Longwood Ave, Boston, MA 02115, USA
^* Present address: Department of Pathology, D4047C, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA

View larger version (60K): [in a new window]   Fig. 1. Amino acid sequence homology between XLMO-2 and the mouse, human and zebrafish homologs. (A) Pile-up analysis of the LMO-2 protein sequences reveals extensive sequence identity between vertebrate LMO-2 proteins. Accession Numbers for sequences used in this comparison: human (hLMO-2, NM_005574; Boehm et al., 1991), mouse (mLMO-2, NM_008505; Boehm et al., 1991) and zebrafish (zLMO-2, AF191560; Thompson et al., 1998). (B) A phylogenetic tree of the LIM-only proteins shows the close relationship of the vertebrate LMO-2 proteins.

View larger version (90K): [in a new window]   Fig. 2. Developmental expression of XLMO-2. RT-PCR analysis on developmentally staged embryos shows that XLMO-2 expression begins after gastrulation and is maintained during embryogenesis. RT-PCR analysis of GATA-1, GATA-2 and XFOG transcripts are included for comparison. Ornithine decarboxylase (ODC) is included as a control of RNA recovery. –RT is a standard reaction that lacks reverse transcriptase and is included as a control for genomic DNA contamination.

View larger version (74K): [in a new window]   Fig. 3. XLMO-2 expression pattern in embryos. RNA in situ hybridization analysis was performed with full-length antisense digoxigenin-labeled probes on albino Xenopus laevis embryos. (A) Stage 21; lateral, dorsal and ventral views of the same embryo showing extensive staining in the nascent ventral blood island (VBI), the tailbud region and the dorsal lateral plate (DLP) mesoderm (black arrowheads). (B) Stage 26; lateral view. Staining is evident in the VBI, DLP (black arrowhead) and tailbud region. Staining is also seen in the brain and in the eye. (C) Stage 33; lateral view. Expression of XLMO-2 is maintained at a high level in the VBI but has diminished in the DLP region (black arrowhead). Staining in the tailbud has condensed to a small region at the tip of the outgrowing tail. (D) Stage 37; lateral view. Staining is evident throughout the circulatory system as XLMO-2 expression is maintained in circulating primitive erythrocytes (asterisked arrowhead). Expression in the tailbud region has condensed to the very tip of the tail. (E) Transverse section of a stage 26 embryo (ventral half only). XLMO-2 stains the ventral blood island (hematopoietic) mesoderm. (F) Transverse section of a stage 21 embryo stained with XLMO-2. Staining is diffuse throughout the mesoderm of the nascent tail bud.

View larger version (134K): [in a new window]   Fig. 4. Comparison of XLMO-2, GATA-1 and SCL expression patterns. RNA in situ hybridization analysis reveals that these genes have very similar expression patterns. Each gene is expressed at a high level in the VBI. GATA-1 expression is restricted to erythrocytes whereas SCL and XLMO-2 are also expressed in other embryonic tissues such as the brain and tailbud.

View larger version (61K): [in a new window]   Fig. 5. Growth factor sensitivity of XLMO-2. Animal pole explants were dissected from embryos injected with various growth factors. Injections were at the 1-cell stage and animal caps were harvested at stage 8. Basic human fibroblast growth factor (bFGF) (20 ng/ml) was applied to uninjected animal caps immediately after dissection. AVg is a chimeric molecule containing the prodomain of activin fused to the functional domain of Vg1. Explants and untreated control siblings were harvested at stage 33. XLMO-2 and GATA-1 transcripts were assayed by semi-quantitative RT-PCR analysis. ODC is ornithine decarboxylase and is used a loading control; –RT is a standard reaction that lacks reverse transcriptase and is included as a control for genomic DNA contamination. WE is a whole-embryo positive control.

View larger version (52K): [in a new window]   Fig. 6. Ectopic expression of XLMO-2 in FGF-treated animal caps induces globin gene expression. (Top) Animal pole explants were harvested from embryos injected with XLMO-2 mRNA, treated with 20 ng/ml bFGF and allowed to develop to stage 33. (Bottom) RT-PCR analysis was performed for tadpole -globin and SCL. XLMO-2 expression in FGF-treated animal caps leads to expression of -globin and SCL. EF-1 was included as a control for RNA recovery. Embryo lane is a whole sibling embryo used as a positive control for the RT-PCR procedure; –RT is a standard reaction that lacks reverse transcriptase and is included as a control for genomic DNA contamination.

View larger version (91K): [in a new window]   Fig. 7. Synergistic activity of LMO-2, SCL and GATA-1 expression in whole embryos. (Top) Embryos were injected at the one cell stage with synthetic mRNAs encoding SCL and LMO-2 (1 ng). Embryos were cultured to stage 33 and stained for tadpole -globin protein by immunohistochemistry. (Bottom) Tadpoles injected with SCL, LMO-2 or GATA-1 alone develop normally and globin expression is limited to the ventral blood island (VBI, black arrows). Combination of SCL and LMO-2 mRNA results in developmental abnormalities. Head structures are diminished or completely missing and globin expression extends laterally around the embryo, reaching at least half way up the body. Co-injection of LMO-2, SCL and GATA-1 results in severe developmental abnormalities: dorsal/anterior structures are absent and globin staining is evident throughout the body axis.

View larger version (40K): [in a new window]   Fig. 8 LMO-2, SCL and GATA-1 act synergistically in animal pole explants to stimulate red blood cell production. (Top) Embryos were injected at the one-cell stage with CMV-driven plasmids encoding LMO-2, SCL and GATA-1 alone or in combination (100 pg). Animal caps were dissected at stage 8, treated with activin (100 pM) and collected for analysis at stage 33. Animal cap cells were enzymatically dissociated, stained with o-dianisidine and collected on glass slides in a cytocentrifuge. Hemoglobinzed cells (erythrocytes), stained brown with the chromogenic reagent, were counted on a bright field microscope. (Graph) Activin treatment of animal pole explants does not result in the formation of red blood cells (mock column, animal pole explants treated with activin from uninjected embryos). Expression of LMO-2, GATA-1 and SCL alone, or in tandem, gives rise to a moderate number of erythroid cells. Co-injection of LMO-2/GATA-1/SCL results in large numbers of o-dianisidine-positive cells in the embryonic explants.