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First published online 2 February 2005
doi: 10.1242/dev.01660

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RBPj-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells

¹ Centre Oncologia Molecular, IDIBELL-Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain
² Department of Immunology and Oncology, Centro Nacional de Biotecnología, CSIC. Darwin, 3. Campus de Cantoblanco, Madrid 28049, Spain

View larger version (91K): [in a new window]   Fig. 1. Expression of Notch family members in the endothelium of the P-Sp/AGM aorta. (A) E9.5 embryo, indicating the site for P-Sp/AGM aorta and hematoxilineosin staining of a transverse section at the indicated level (100x) (B,C,D) Whole-mount of (B) Notch receptors, (C) Notch-ligands and (D) Notchtarget genes and transverse sections of E9.5 and 10.5 aortas. (B) Notch1 is expressed in few scattered cells at E9.5 and these cells increase at E10.5. Notch2 and Notch3 are not expressed in the aorta. Notch4 shows a homogenous staining pattern in most of the cells of the endothelium at E9.5 and 10.5. (C) Dll1 and Dll3 are not expressed in the aorta. Jag1, Jag2 and Dll4 are expressed in few scattered cells at E9.5 and these cells increase at E10.5 (D) Hes1 is not expressed at E9.5 but shows expression at E10.5 in cells budding from the endothelium. Hrt1/herp2 is expressed in the ventral endothelium and hematopoietic clusters at E9.5 and 10.5. Hrt2/Herp1 shows more diffused expression at E9.5 and ventral endothelium at E10.5. Orientation of the aortas is dorsal (up) to ventral (down).

View larger version (81K): [in a new window]   Fig. 3. Absence of hematopoietic cells and increase of endothelial cells in the P-Sp/AGM of RBPj mutant embryos. (A) Semiquantitative RT-PCR analysis in dissected P-Sp of E9.5 wild-type and RBPj mutants. Representative PCR products after 35 and 40 cycles of two independent experiments are shown (B) WISH of hematopoietic transcription factors and transverse sections of E9.5 aortas of wild-type and RBPj mutants. (C) WISH with VE-C (upper panel), expression of VE-C in the aorta (middle panels) and expression of PECAM/CD31 by immunofluorescence (lower panel) on transverse sections of E9.5 aortas of wild-type and RBPj mutants. Orientation of the aortas is dorsal (up) to ventral (down).

View larger version (62K): [in a new window]   Fig. 2. Intra-embryonic hematopoiesis is impaired in the RBPj mutant embryos and they display an aberrant expression of Notch family members in the aorta (A) Semiquantitative RT-PCR analysis in dissected P-Sp of E9.5 wild-type and RBPj mutants. Representative PCR products after 35 and 40 cycles of two independent experiments are shown. (B) WISH, with the indicated probes and transverse sections of E9.5 aortas of RBPj mutants. Orientation of the aortas is dorsal (up) to ventral (down). (C) Hematopoietic CFC from dissected P-Sp of E9.5 wild-type and RBPj mutants. Bars represent the average number of CFCs and standard deviation from three different embryos. (D) Liquid cultures with IL3 and SCF-conditioned media from P-Sp of E9.5 wild-type and RBPj mutants at days 0 and 6. (E) After 6 days in culture, cells were assayed for the expression of CD45 by flow cytometry and (F) the number of CFCs generated. Bars represent the average number of CFCs obtained from one wild-type embryo and pools of two or three mutant embryos equivalent (e.e.) in three independent different experiments.

View larger version (55K): [in a new window]   Fig. 4. Notch1/RBPj regulates Gata2 transcriptional activity. (A) Semiquantitative RT-PCR analysis of Gata2, Aml1 and Scl expression in 32D wild-type or N1IC-expressing cells. Representative PCR products after 35 and 40 cycles of two independent experiments are shown. Quantitated relative mRNA levels of Gata2, Aml1 and Scl are shown in the lower graph. (B) Chromatin immunoprecipitation with anti-N1 from 32D wild-type cells and 32D-N1IC cells. PCR detection of the Gata2, Aml1 and Scl, Hes1 and ß-globin promoters from the precipitates is shown. (C) Double in-situ hybridization with Gata2 and Notch1 on transverse section of wild-type E10.5 aortas. (D) Section of WISH that shows Hes1 expression in hematopoietic clusters budding from the aorta from E10.5. (E) Chromatin immunoprecipitation with anti-N1 (-N1) from wild-type and RBPj mutant whole E9.5 embryos. PCR detection of the Gata2 and Aml1 promoter is shown.

View larger version (54K): [in a new window]   Fig. 5. Notch1+/Gata2+ cells in the P-Sp/AGM endothelium are Jag1+/Jag2-. Double in-situ hybridization on transverse section of wild-type E10.5 aortas. (A) Hybridization of Notch1 with Jag1 (upper), Jag2 (middle) and Dll4 (lower panels). Representative photographs of at least three hybridizations are shown (B) Model for Notch function in the formation of hematopoietic clusters from the aorta endothelium during development.

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