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Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish

Eric C. Liao*,{dagger}, Nikolaus S. Trede*, David Ransom*, Augustin Zapata{ddagger}, Mark Kieran* and Leonard I. Zon*,§

* Division of Hematology/Oncology, Children’s Hospital, Department of Pediatrics and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
{dagger} Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
{ddagger} Department of Cell Biology, Complutense University, 28040 Madrid, Spain



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  		Fig. 1. The bls mutation causes severe embryonic anemia that recovers in the larval stage. Wild-type and bls embryos stained with o-dianisidine, which detects cells containing hemoglobin. All embryos in this and subsequent figures are shown in lateral views, oriented with anterior and dorsal towards left and top of the page, respectively, unless otherwise specified. Blood cells pool in the Duct of Cuvier of wild-type embryos (arrowhead) at 36 hpf, but are absent in bls mutants. However, as the bloodless embryos develop beyond 5 dpf, blood cells begin to accumulate. By 7.5 dpf, blood cells can be detected near wild-type levels in the bls mutants, as they circulate through the heart and branchial vessels (arrowhead).

 


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  		Fig. 2. Disruption of primitive hematopoiesis in bls mutants. In situ hybridization analysis of hematopoietic markers scl, gata1, gata2, ikaros and the endothelial marker flk1 in bls mutants. (A) Expression of scl (red) and gata1 (blue) in wild-type and bls embryos. Cells expressing both scl and gata1 appear purple. At eight somites, scl transcripts can be detected in the anterior and posterior lateral plate mesoderm. The expression of scl in the anterior lateral plate does not appear to be perturbed in bls. By contrast, the expression of scl and gata1 in bls is decreased at eight somites, with scl expression appearing less reduced than that of gata1 (arrowhead). By 18 somites, scl expression persists in the ICM but gata1 expression cannot be detected. At 23 hpf, some scl transcripts can be detected in the posterior ICM (arrowhead with asterisk) and gata1 expression remains absent. Note that the scl/gata1 double in situ clearly defines the anterior versus posterior ICM, as the anterior ICM appears dark purple and the posterior ICM appears red. (B) Expression of gata1 (in dark purple) and myosin (in blue) in wild-type and bls embryos. In wild-types, gata1 expression is shown at eight somites in the lateral plate mesoderm (arrowhead). Myosin is expressed in the differentiating somites. gata1-expressing cells in the lateral plate mesoderm converge towards the midline by 18 somites to form the ICM, which expresses gata1 (arrowhead). Expression of gata1 persists in the hematopoietic progenitors at 23 hpf. By contrast, gata1 is absent in bls embryos, from as early as eight somite stage. (C) Expression of gata2, ikaros, and flk1 in wild-type and bls embryos. gata2 transcripts are detectable in bls, although the experession appears to be reduced (arroheads and arrowheads with asterisks), whereas ikaros expression is absent in bls. By contrast, flk1 expression is undisturbed by bls.

 


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  		Fig. 3. Apoptosis in the ICM and cells of the dorsal trunk and tailbud. (A) Acridine Orange staining of wild-type and bls embryos at 15- somites and 23 hpf detects cells undergoing apoptosis. Images of embryos under light-microscopy (far left and far right) are shown as anatomic references for the fluorescence images (middle left and right). Apoptotic cells appear as yellow dots when stained with Acridine Orange. Low level apoptosis is can be seen in wild-type embryos (sparse yellow dots). However, bls embryos exhibit marked apoptosis in the ICM (arrowhead) and dorsal trunk mesenchyme (arrowheads with asterisk). The numerous apoptotic cells line the dorsal trunk of the bls embryo, and extend around the tailbud into the ICM. (B) Transcripts of spt can be detected in the ICM at 23 hpf in wild-type but not bls embryos (arrowhead). Expression of spt in the tailbud and spinal neurons are unperturbed by bls. Likewise, tbx6 expression is absent in the ICM of bls mutants, but is normally expressed in the tailbud. Neural crest derived Rohon-Beard cells are detected with HNK-1 antibody along the dorsal trunk of both wild-type and bls embryos (arrowhead). Axial mesoderm markers ntl and shh exhibit wild-type expression patterns in bls notochord and tailbud.

 


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  		Fig. 4. Primitive macrophages develop normally in bls. Primitive macrophages develop from anterior paraxial mesoderm, and express (A) pu.1, (B) cmyb and (C) dra. Expression of pu.1 is unique to primitive macrophages, whereas cmyb and dra transcripts are also detected in the ICM at 20 somites. (A) Expression of pu.1 is not perturbed by bls, and can be detected in the anterior paraxial mesoderm at 12 somites. (B) cmyb expression in the primitive macrophage is unaffected by bls (arrowhead) but its expression in the ICM (asterisk) is absent at 20 somites. (C) Similarly, dra expression in the primitive macrophage primordia is unaffected by bls. However, dra expression in bls is significantly reduced in the lateral plate mesoderm at 12 somites (asterisk) and in the ICM at 20 somites, similar to the pattern of scl expression.

 


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  		Fig. 5. Initiation of lymphopoiesis is delayed in bls. Lymphoid precursors expressing rag1 can be detected in the thymus around 4 dpf. (A) Lymphoid precursors are present in the thymus of wild-type embryos at 4.5 dpf (arrowhead), but are absent from bls. However, if the bls embryos are raised to 7.5 dpf, rag1 expression begins to appear in the thymus, indicating the appearance of lymphoid precursors (arrowhead with asterisk). (B) Coronal section of 4.5 dpf embryos shows that lymphoid cells cluster in the thymus of wild-type embryos (arrowhead), but are absent in the bls thymus. Corresponding to the recovery of rag1 expression at 7.5 dpf, lymphocytes of wild-type morphology can be detected in the thymus of bls embryos (double arrowheads).

 


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  		Fig. 6. Overexpression of bmp4, scl and gata1 in bls and clo mutants. Plasmid construct with CMV promoter driving (A) GFP, (B) bmp4, (C) scl and (D) gata1 were micro-injected into wild-type, bls and clo embryos. The injected embryos were fixed at 23 hpf and analyzed by scl, gata1 or globin in situ hybridization. (A) Plasmid expression of GFP did not perturb embryo development and did not alter the expression of scl or gata1 when compared with uninjected embryos. Expression of scl can normally be detected in the posterior ICM of bls embryos (arrowhead), and in 5-10 cells of the wedge region in the anterior ICM of clo embryos (arrowhead with asterisk). (B) Wild-type embryos injected with bmp4 were ventralized, with an expanded ICM that expresses scl and gata1. The arrowhead indicates the wedge region of anterior ICM. Ventralized bls embryos lacked scl- or gata1-expressing cells in the anterior ICM, but the number of scl-positive cells in the posterior ICM was slightly increased (arrowhead with asterisk). Ventralized clo embryos did not express scl or gata1. (C) Four scl injected bls embryos are shown (rsc1-4), each exhibiting representative levels of partial rescue of hematopoietic progenitors (arrowheads). The hematopoietic rescue in scl injected clo embryos was more pronounced (arrowhead with asterisk). (D) Overexpression of gata1 led to normal level of globin transcripts in the anterior ICM of wild-type embryos, but no globin expression was detected in the bls mutants.

 


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  		Fig. 7. Non-cell autonomous requirement for bls in primitive hematopoiesis. Donor cells labeled with biotin-dextran were transplanted into unlabeled hosts at the sphere stage, to determine whether donor cells can contribute to blood in the host environment. The resulting chimeric animals were analyzed for gata1 expression. Genotype of the host is indicated outside, and that of the donor within, the parenthesis (host genotype -> (donor genotype)). Donor-derived cells were detected by avidine-peroxidase color reaction, and appear brick red. Cells containing gata1 transcripts appear blue. Donor cells that contribute to blood will be colored with red and blue, appearing dark purple (arrowheads).

 

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© The Company of Biologists Ltd 2002