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First published online June 8, 2005
doi: 10.1242/10.1242/dev.01884

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A Pbx1-dependent genetic and transcriptional network regulates spleen ontogeny

¹ Department of Cell and Developmental Biology, Cornell University, Weill Medical School, New York, NY, 10021, USA
² Dipartimento di Biologia Animale, Universita' di Modena e Reggio Emilia, Via Università 4, 41100, Modena, Italy
³ Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA
⁴ The Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, University of Toronto, Toronto, Ontario M5G 1X5, Canada
⁵ Università Vita-Salute San Raffaele, 20132 Milan, Italy
⁶ Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (109K): [in a new window]   Fig. 1. Agenesis of the spleen in Pbx1-/- embryos. (A,B) Comparative whole-mount preparation of upper abdominal organs at E15.5 shows the lack of a spleen, together with liver hypoplasia, in Pbx1-/- embryos (B). The spleen forms lateral to the stomach, and appears as a reddish ribbon-shaped organ in wild-type embryos (black dashes in A). (C,D) Histology of Hematoxylin and Eosin-stained transverse sections at E15.5 show agenesis of the spleen in Pbx1-/- embryos compared with wild-type littermates. (E,F) Histology of Hematoxylin and Eosin-stained transverse sections of E13.5 wild-type and Pbx1-/- embryos. A visible spleen primordium forms as a mesenchymal condensation within the dorsal mesogastrium (Dm, arrows) in wild-type embryos (E). In Pbx1-/- littermates (F), no mesenchymal condensation is detectable within the Dm (arrowheads). (G-J) Histology of Hematoxylin-stained transverse sections of wild-type and Pbx1-/- embryos in the region of the stomach enlargement at E10-10.5. The splanchnic mesoderm lateral to the stomach enlargement (G,I) consists of a thick epithelial-like plate of cells (arrows) that encloses unorganized mesenchyme. The black boxes indicate the regions magnified (40x) in H,J, which highlight the organized epithelial-like cellular structure of the splanchnic mesoderm. C, coelomic cavity; Dm, dorsal mesogastrium; L, liver; Sp, spleen; St, stomach.

View larger version (126K): [in a new window]   Fig. 2. Perturbed expression of Hox11 and Wt1, known regulators of spleen development, in Pbx1-/- embryos. Transcripts for the spleen regulators Hox11, Wt1, Pod1 and Nkx3.2 were detected by in situ hybridization to frozen sagittal sections of wild-type and Pbx1-/- upper abdominal organs at E11-11.5 and E12-12.5, as indicated. The splenic primordium is outlined by black dashes. (A-D) Both at E11-11.5 and E12-12.5 Hox11 expression is absent in the spleen primordia of Pbx1-/- embryos, compared with wild-type littermates. (E-H) Wt1 expression is also absent in spleen anlage of Pbx1-/- embryos compared with wild-type littermates both at E11-11.5 and E12-12.5. It is noteworthy that Wt1 expression is still present in the mesothelial lining that surrounds the mesenchyme of the splenic anlage (arrow). (I-L) Pod1 and (M-P) Nkx3.2 are normally expressed within the developing spleen primordia of Pbx1-/- embryos compared with wild-type littermates, both at E11-11.5 and E12-12.5. L, liver; Sp, spleen; St, stomach.

View larger version (78K): [in a new window]   Fig. 3. Analysis of Pbx1 and Nkx2.5 gene expression during early spleen organogenesis. In situ hybridization was performed on frozen sagittal sections of E12-12.5 embryonic upper abdominal organs. (A-F) Normal expression of Pbx1 within the developing splenic anlage of Hox11-/-, Pod1-/- and Nkx3.2-/- embryos. (G-L) Perturbed expression of Nkx2.5 in Pod1-/- and Pbx1-/- embryos. Nkx2.5 expression is unperturbed in the developing splenic anlage of Hox11-/- embryos (H), while it is absent in Pod1-/- (J) and Pbx1-/- (L) embryos, compared with their wild-type littermates (G,I,K). L, liver; Sp, spleen; St, stomach.

View larger version (74K): [in a new window]   Fig. 4. Genetic interaction of Pbx1 and Hox11 during spleen organogenesis. (A-D) Immunostaining of sagittal sections of E11.5 (A,B) and E 12.5 (C,D) Hox11lacZ/+ embryonic upper abdominal organs. Arrowheads in A-D indicate cells expressing Pbx1 but not Hox11. Pbx1 is visualized in the splenic anlage with DAB (brown staining), and Hox11 by ß-galactosidase staining (blue). (B,D) Enlargements of the black rectangle depicted on the splenic anlage in A,C. Arrows in B,D indicate cells in which Pbx1 and Hox11 colocalize. (E,F) The spectrum of malformations of double heterozygous spleens (F) is compared with the normal gross morphology of Pbx1+/- spleens (E). The Pbx1+/-;Hox11+/- double heterozygous spleens are hypoplastic (F), and display sickle shapes, indentations, tubercles and nodules, as well as fusions of two spleens (arrowheads). All spleens were isolated from 6- to 8-week-old mice. (G,H) Hematoxylin and Eosin-stained spleen sections reveal no abnormalities in splenic structure of Pbx1+/-;Hox11+/- double heterozygous mice. Distribution of white (arrows) and red (arrowheads) pulp is normal within the spleen parenchyma of double heterozygous mice (H) compared with Pbx1+/- (not shown), Hox11+/- (not shown) and wild-type (G) littermates. (I,J) PNA-stained spleen sections of mice immunized with sheep red blood cells (SRBC). Formation of germinal centers (GC; arrows), as indicated by PNA staining (brown), appears normal in Pbx1+/-;Hox11+/- double heterozygous mice (J) compared with Pbx1+/- (not shown), Hox11+/- (not shown) and wild-type (I) littermate controls. L, liver; Sp, spleen; St, stomach.

View larger version (63K): [in a new window]   Fig. 5. Similar reduction of spleen progenitor cell proliferation in Pbx1-/- and Hox11-/- embryos. (A,C) BrdU in vivo labeling of sagittal sections of upper abdominal organs shows a striking difference in the percentage of BrdU-positive nuclei (brown) within the spleen anlage of Pbx1-/- and Hox11-/- embryos, compared with wild-type littermates at E13.5. (B,D) Proliferation of spleen mesenchymal progenitors is reduced by 50% in Pbx1-/- and Hox11-/- embryos, respectively, when compared with wild-type littermates. The results are expressed as total BrdU-positive cells per mm2 of spleen anlage. Data are mean±s.e.m. of four E 13.5 embryos analyzed for each genotype. Black bars, wild type; grey bars, Pbx1-/- and Hox11-/- embryos; Sp, spleen; St, stomach.

View larger version (52K): [in a new window]   Fig. 6. Recruitment of Pbx1 and Hox11 to the mouse Hox11 promoter. (A) Schematic illustration of 1.2 kb of the Hox11 genomic segment with known promoter activity that contains Pbx1-binding sites, and a 5' upstream region. Primers used for PCR analysis are indicated by arrows (each pair in a different color) and the oligoprobe (PX1) used for EMSA is indicated by a black box. (B) Binding of Pbx1 and Hox11 to the (PX1) oligo within the Hox11 promoter. Nuclear extracts derived from wild-type primary embryonic spleen cells were subjected to EMSA with a radiolabeled PX1 probe containing a Pbx1 wild-type (PX1) or mutated (mPX1) core binding site (underlined), as indicated above gel lanes. Asterisk indicates non-specific band. (C) Primary cells isolated from embryonic spleen stained in culture for the mesodermal marker smooth muscle actin (green fluorescence). Western blot analysis (panel below) demonstrates that these cells produce Pbx1, Prep1 and Hox11 proteins. Two isoforms of Hox11 are present in embryonic spleen, as indicated (Yamamoto et al., 1995). (D,E) For ChIP analysis, chromatin was subjected to immunoprecipitation (IP) using antibodies specific for Pbx1b (anti-Pbx1b) (D) or Hox11 (anti-Hox11) (E). As negative controls, IPs were also performed with an anti-GFP antibody, rabbit serum (RS) or no antibody (No Ab). A primer pair that amplifies a region within the Bmp4 promoter was used as an additional negative control. (F) Synergistic activation of the Hox11 promoter by Pbx1, Prep1 and Hox11 proteins. Luciferase activity was assayed from transiently transfected NIH 3T3 cells. Co-transfection assays were performed in the presence (+) of the indicated expression vectors encoding Pbx1, Prep1 or Hox11, and with a vector containing the promoter regulatory region of Hox11 (p540), or with vector alone (pGL2). Data are expressed as the fold activation over the p540 basal luciferase activity. Bars represent the mean of three independent transfections (performed in duplicate)±s.e.m. normalized for ß-galactosidase activity (internal control) within each experiment.

View larger version (35K): [in a new window]   Fig. 7. Pbx1-dependent genetic network and transcriptional pathway regulate spleen ontogeny. (A) Transverse section of abdominal organs schematically illustrates the location of the spleen primordium during vertebrate development. Adapted, with permission, from Sadler (Sadler, 1995). To the right, the Pbx1-Hox11 transcriptional hierarchy regulating spleen ontogeny is depicted, with the Pbx1-dependent pathway in red. The Nkx2.5 downstream pathway is illustrated with a broken arrow as the requirement for Nkx2.5, an early marker of splenic progenitor cells, has not yet been demonstrated in spleen development. (B) Within the Pbx1-Hox11 transcriptional pathway, Pbx1 directly regulates Hox11 in spleen progenitor cells and Hox11, in turn, regulates its own promoter together with Pbx1.

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