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First published online 5 November 2003
doi: 10.1242/dev.00875

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Lmo2 and Scl/Tal1 convert non-axial mesoderm into haemangioblasts which differentiate into endothelial cells in the absence of Gata1

Martin Gering¹, Yoshihiro Yamada^2,*, Terence H. Rabbitts² and Roger K. Patient^1,

¹ Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK
² Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (83K): [in a new window]   Fig. 1. Lmo2 and Scl/Tal1 induce early blood and endothelial cells throughout the lateral mesoderm. (Aa-i,Ba-b,Ca-b) Flat mounts; anterior top. (Cc-h) Transverse sections; dorsal top. (D) Schematic of results. (A) In uninjected embryos (a-c), lmo2end. (a), scl/tal1end. (b) and fli1 (c) expression overlap in bilateral stripes in the ALM and the PLM. fli1 is also expressed in anterior neural crest cells (c, asterisk). [The nkx2.5+ (see Fig. 2A) cardiogenic mesoderm is located in the gap between the ALM and the PLM.] (A) In scl/tal1-injected embryos (d-f), lmo2end. (d), scl/tal1end. (e) and fli1 (f) are ectopically expressed in the SPM (arrows). (A) In scl/tal1-lmo2-injected embryos (g-i), lmo2end. (g), scl/tal1end. (h) and fli1 (i) are, in addition, ectopically expressed in a broader domain in the head (arrowheads) and in the cardiogenic mesoderm (brackets). (B) Lineage tracing reveals that ectopic scl/tal1end. (b) is induced on the injected, ß-galactosidase-positive side of the embryo (a). (C) Sections (taken at the levels indicated by c-h, shown below) confirm that scl/tal1-lmo2 injection induced scl/tal1end. expression in the head mesoderm (c,d), in the cardiogenic mesoderm (e,f), as well as in the SPM (g,h). (D) Whereas scl/tal1 injection (s-inj.) induces ectopic haemangioblasts (purple) in the somitic mesoderm, scl/tal1-lmo2 injection (sl-inj.) expands haemangioblasts into the head, heart (H) and somitic mesoderm. Not, notochord.

View larger version (108K): [in a new window]   Fig. 2. The induction of early blood and endothelial progenitors occurs at the expense of the myocardial fate. (A-C and H,I) Flat mounts of embryos; anterior, top. (D-G and J,K) Anterior views of whole mounts; ventral, bottom. (L,M) Lateral views, anterior to the left. (A-C) At the 10-somite stage, nkx2.5 expression is reduced on the injected side in scl/tal1-lmo2 (SL)-injected embryos (C, arrow) but is perfectly normal in scl/tal1 (S)-injected (B) embryos. (D I) Likewise, gata4 (D,E), gata6 (F,G) and tbx5.1 (H,I) were reduced (E,G,I, arrows) on the side injected with scl/tal1-lmo2. (J-M) vmhc+ (Yelon et al., 1999) ventricular tissue was reduced at the 18-somite stage (J,K) and the nkx2.5+ heart tissue was reduced by 34 hpf (L,M) by injection with scl/tal1-lmo2.

View larger version (64K): [in a new window]   Fig. 3. scl/tal1-lmo2 coinjection induces erythropoiesis more anteriorly at the expense of the pronephros. (AE) Flat mounts. (F,G) Dorsal views of whole mounts; anterior, top. (H) Schematic of results. (A,B) scl/tal1-lmo2 injection (SL) increased the number of ßE1+ erythroid cells anteriorly and laterally, but they did not fuse at the midline. ßE1 was induced in non-neural ectoderm (B, asterisk). (C,D) In scl/tal1-lmo2-injected embryos, gata1 expression (purple) is broader and expanded anteriorly adjacent to somites 1-5 (arrows). (E-H) Scl/Tal1 and Lmo2 reduced expression of the pronephros (PN) marker wt1 (G), suggesting that erythroid development is expanded at the expense of the pronephros as shown in H.

View larger version (76K): [in a new window]   Fig. 4. Lateral expansion of erythroid development at the expense of the pronephric duct. All embryos flat mounted; anterior, top. (A,B) In uninjected embryos, gata1 expression pattern (purple) overlapped with the stronger medial aspect of fli1 expression (red) in the PLM (blue arrow in b). (C,D) In scl/tal1-lmo2-injected embryos (SL), gata1 was excluded from the fli1+ cells in the head (green arrow), heart (bracket) and somitic mesoderm (black arrow) but completely overlapped with fli1 expression in the PLM (blue arrows), demonstrating expansion into the weaker, lateral aspect of the fli1 domain. (E,F) In uninjected embryos, the weaker lateral aspect of the fli1 domain overlaps with expression of the PND marker pax2.1. Yellow arrows in A,B,C,E indicate fli1+ PND progenitors. (G,H) pax2.1 expression was reduced in the PLM of scl/tal1-lmo2-injected embryos (H). (I) Schematic showing that in scl/tal1-lmo2-injected embryos, gata1+ erythroid cells (red) form ectopically from haemangioblasts developing from PLM that normally gives rise to the PND (yellow). Ectopic haemangioblasts induced in the head, heart and somitic mesoderm (purple) do not develop into erythrocytes, showing that erythropoiesis is regionally restricted.

View larger version (78K): [in a new window]   Fig. 5. Gata1-scl/tal1-lmo2 injection induced anterior erythropoiesis without causing overt ventralisation/posteriorisation. (A-D,G-J) Whole mounts; anterior, left in A-D, top in G-J. (E,F) Flat mounts; anterior, left. (A-D) alas2 expression was restricted to posterior regions in uninjected (A) and gata1-scl/tal1-injected (G1S, C) embryos. gata1-scl/tal1-lmo2 injection (G1SL, B) induced alas2 expression in the head (black arrowheads) (A-C) Red arrows mark pax2.1 expression in the optic stalk and in the midbrain-hindbrain boundary in the head. (D) Equimolar amounts of gata2 mRNA (G2SL) could not replace gata1. (E,F) gata1-scl/tal1-lmo2 injection (F) induced anterior expression of gata1end.. (G,H) gata1-scl/tal1-lmo2 injection (25 pg of each mRNA) induced anterior erythropoiesis without inducing bmp4. (I,J) At 100 pg, the three mRNAs delayed development and bmp4 expression was maintained longer ventrally but not induced dorsally. Arrowheads in J indicate the ventral side of the embryo.

View larger version (95K): [in a new window]   Fig. 6. The number of myeloid cells is not increased in scl/tal1-lmo2-injected embryos. (A-H) Flat mounts; anterior, top. (I,J) Anterior views of whole mounts with dorsal towards the top. (A-H) Some but not all myeloid genes were ectopically activated in scl/tal1-lmo2-injected (SL inj.) embryos. Arrowheads indicate gene expression in anterior myeloid progenitors (A-H). The orange arrow indicates ectopic pu.1 expression around the tailbud (B). Red arrows indicate runx1+ Rohon-Beard sensory neurons (C). Brackets indicate ectopic gene expression in the heart region (F,H). Black arrows indicate normal gene expression in the PLM (A-C,E,G). Blue arrows indicate the anterior gene expression limit in the PLM in scl/tal1-lmo2-injected embryos (D,F,H). Purple arrows indicate laterally expanded gene expression in the PLM (D,F,H). White arrow indicates ectopic runx1 expression in the anterior SPM (D). Green arrows indicate the expansion of dra+ cells in the posterior SPM (G,H). (I,J) The number of mature L-plastin+ myeloid cells was not increased in scl/tal1-lmo2-injected embryos.

View larger version (108K): [in a new window]   Fig. 7. The number of endothelial progenitors is increased in scl/tal1-lmo2-injected embryos. (A-F,J-M) flat mounts. Anterior, top in A-F, left in J-K. (G-I) Whole mounts; anterior, left. (N) Schematic of results. (A-F) In scl/tal1-injected (S inj.) 10 somite (14 hpf) embryos, the endothelial genes flk1 and flt4 were ectopically expressed only in the SPM (B,E; arrows). In scl/tal1-lmo2-injected (SL inj.) embryos, flk1 showed strong ectopic expression in the head (C, arrowhead) and in the heart region (C, bracket), while flt4 was expressed in a wider domain in the head (F, arrowhead) that was expanded posteriorly into the heart region, leaving a smaller gap between the anterior and posterior flt4+ domains (F, bracket). (G-I) At 22 hpf, the late endothelial marker tie2 was expressed in all ECs including the cells of the dorsal aorta (DA) and axial vein (AV, G). Tie2 was ectopically expressed only in the posterior of scl/tal1-injected (S) embryos (H, arrows), but all along the anteroposterior axis in scl/tal1-lmo2-injected embryos (I, arrows and arrowheads). (J-M) Most of the tie2+ cells were scl/tal1end. negative, suggesting that they were differentiating ECs. tie2 (J) was normally expressed in all ECs of the axial vessels (DA and AV) and the head (black arrows), scl/tal1 (L) is expressed in erythrocytes (red arrow), blood and endothelial progenitors of the tail (purple arrow), ECs of the head (black arrows) and the cardinal veins (green arrows), as well as in ventral neurons of the spinal cord (blue arrow). Ectopic expression of tie2 was widespread anteriorly (K, arrowhead), but scl/tal1end. expression was limited to its normal expression in bilateral patches in the head mesoderm (L,M, black arrows). In the trunk, scl/tal1end. was expressed in ventrolateral cells (M, red arrows) similar to the ßE1+ blood cells. By contrast, tie2 expression was found medially (K, red arrow) as well as dorsally (I, black arrows). Only posteriorly, did co-expression mark early blood and endothelial progenitors (I,K,M, brackets). (N) In the absence of a blood-inducing intrinsic/extrinsic factor, Scl/Tal1- and Scl/Tal1-Lmo2-induced ectopic haemangioblasts in the head, heart and somitic mesoderm express flk1 and flt4 (blue) and develop into ECs, suggesting that endothelial development is the default state.