The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis - PubMed (original) (raw)

The T cell leukemia LIM protein Lmo2 is necessary for adult mouse hematopoiesis

Y Yamada et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The LIM-finger protein Lmo2, which is activated in T cell leukemias by chromosomal translocations, is required for yolk sac erythropoiesis. Because Lmo2 null mutant mice die at embryonic day 9-10, it prevents an assessment of a role in other stages of hematopoiesis. We have now studied the hematopoietic contribution of homozygous mutant Lmo2 -/- mouse embryonic stem cells and found that Lmo2 -/- cells do not contribute to any hematopoietic lineage in adult chimeric mice, but reintroduction of an Lmo2-expression vector rescues the ability of Lmo2 null embryonic stem cells to contribute to all lineages tested. This disruption of hematopoiesis probably occurs because interaction of Lmo2 protein with factors such as Tal1/Scl is precluded. Thus, Lmo2 is necessary for early stages of hematopoiesis, and the Lmo2 master gene encodes a protein that has a central and crucial role in the hematopoietic development.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Requirement for Lmo2 expression in hematopoiesis in vitro. ES cells were differentiated in the presence of interleukin 1α, stem cell factor, interleukin 3, and erythropoietin, and PCR amplifications were performed with cDNA synthesized from RNA isolated from embryoid bodies at day 0 and at 4 and 10 days after induction, with gene-specific primers for the indicated transcripts. Actin was used as a quality control for the RNA prepared from ES cells. ES cells examined were wild-type (+/+), Lmo2 −/−, and Lmo2 −/− into which an Lmo2 expression vector had been transfected (Lmo2 −/−R). Sizes of the transcripts are indicated.

Figure 2

Figure 2

Lmo2 −/− ES cells cannot differentiate into hematopoietic tissues in vivo. (A) Tissue contribution of Lmo2 +/− and −/− ES cells in chimeric mice. Tissue extracts from chimeric mice were prepared and the relative proportion of GPI isozymes 1B (C57BL/6 blastocysts) or 1A (129 ES cells) was assayed. Chimeric mice were generated by injecting Lmo2 +/− ES cells (C320) or two Lmo2 −/− ES cell clones, 71(−/−) and 53(−/−). (B) PCR amplifications were performed with the polymorphic microsatellite marker D10Mit180 (15) and DNA isolated from tissues of chimeric mice. PCR products differ between C57BL/6 (blastocysts) and 129 (ES cells), as seen in products from a 50:50 mixture of C57BL/6 and 129 DNA.

Figure 3

Figure 3

Contribution of the Lmo2 +/− (C320) and −/− (clones 71 and 53) ES cells to tissues in chimeric mice. Individual mice are indicated and the approximate coat color chimerism is given. The mouse tissue was assayed with combinations of techniques. In each case, GPI analysis was first performed and these data were confirmed by the microsatellite PCR method (complete concordance was obtained). For C320 thymus sample E, no GPI data is available, the results being obtained from microsatellite and Y chromosome PCR only. Blood (∗) analysis was carried out by the modified globin analysis (13) and was only performed on C320 chimeras A, E, D, F, N, P, R, and S; clone 71 chimeras D, E, F, G, H, and J; and clone 53 chimera A. Boxes: green, ES contribution was detectable; red, no ES contribution was detectable; white, no information. Data from those with more than 50% coat color chimerism are shown. THY, thymic cells; SPN, splenic cells; MØ, peritoneal macrophage; KID, kidney; HRT, heart; LIV, liver; BRN, brain.

Figure 4

Figure 4

Hematopoietic rescue by reexpression of Lmo2 −/− ES cells. (A) PCR amplifications were performed with Y chromosome-specific primers (giving the Uty product) and DNA isolated from 24-h cultures of peritoneal macrophages from female chimeric mice produced with the Lmo2 −/− ES clone 71 (mice 71H and 71J) or the BR12 _Lmo2_-expressing −/− clone (−/−R; mouse 12H). The PCR products were separated on 4% Nusieve agarose. DNA quality for each PCR was tested simultaneously with the microsatellite primer pair D10Mit180 (data not shown). Electrophoresis of control PCRs using DNA samples from a male mouse and from ES cells are also shown. (B) Modified hemoglobin analysis of blood samples (13) of Lmo2 −/− clone 71 (71E and 71F) and clone BR12 (12F, 12G, and 12H) chimeric mice. Hemoglobins were separated on cellulose acetate thin-layer plates. Hbbs is specific for C57BL/6 (blastocysts) and Hbbd is specific for 129 (ES cells) as indicated by arrows. Note the relatively faint ES cell-specific bands seen in BR12 samples; this phenomenon was also observed in the Tal1/Scl rescue experiments (20) and is possibly due to the lack of proper regulation of gene expression (e.g., perhaps a promoter efficiency effect or a chromosome location effect on Lmo2 expression).

Figure 5

Figure 5

Rescues in all components of hematopoiesis by Lmo2 reconstitution. (A) Similar PCR analyses were performed, as in Fig. 4, with DNA from purified splenic cells and purified thymic cells, and the products were fractionated on 4% Nusieve agarose gels. Three chimeras were analyzed from the Lmo2 −/− ES clone 71 (71A, 71H, and 71J) and one chimera made from ES clone BR12 (−/−R; 12C). (B) Summary of ES cell contribution of the Lmo2 −/− clones in which Lmo2 expression has been restored by using an Lmo2 expression vector (BR3 and BR12). Individual chimeric mice were analyzed, as shown, and the sex and estimated level of coat color chimerism are listed. Green boxes indicate ES contribution was detectable and the method applied is shown (M, microsatellite PCR; g, globin; G, GPI; Y, Y chromosome PCR). White boxes indicate no ES contribution was detectable. THY, thymic cells; SPN, splenic cells; MØ, peritoneal macrophage; KID, kidney; HRT, heart; LIV, liver; BRN, brain.

Similar articles

Cited by

References

    1. Rabbitts T H. Nature (London) 1994;372:143–149. - PubMed
    1. Boehm T, Foroni L, Kaneko Y, Perutz M P, Rabbitts T H. Proc Natl Acad Sci USA. 1991;88:4367–4371. - PMC - PubMed
    1. Royer-Pokora B, Loos U, Ludwig W-D. Oncogene. 1991;6:1887–1893. - PubMed
    1. Larson R C, Osada H, Larson T A, Lavenir I, Rabbitts T H. Oncogene. 1995;11:853–862. - PubMed
    1. Larson R C, Lavenir I, Larson T A, Baer R, Warren A J, Wadman I, Nottage K, Rabbitts T H. EMBO J. 1996;15:1021–1027. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources