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First published online 25 August 2004
doi: 10.1242/dev.01364

Development 131, 4665-4675 (2004)
Published by The Company of Biologists 2004
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The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2

Jacob Hecksher-Sørensen1,*,{dagger}, Robert P. Watson1,*, Laura A. Lettice1, Palle Serup2, Lorraine Eley3, Carlo De Angelis1, Ulf Ahlgren1,{ddagger} and Robert E. Hill1,§

1 Comparative and Developmental Genetics Section, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
2 Department of Developmental Biology, Hagedorn Research Institute, Niels Steensens Vej 6, 2820 Gentofte, Denmark
3 The Institute of Human Genetics, The International Centre for Life, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK



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  		Fig. 1. Advent of the spleno-pancreatic mesenchyme and the relationship to the SMP. (A) Reconstruction of an E10.5 embryo following analysis by optical projection tomography (OPT). Yellow and red boxes indicate the planes of the sections shown in B and C. (B,C) Cross-sections through an E10.5 embryo in the region of the stomach (B) and dorsal pancreas (C). Boxed region in C indicates the embryonic position of the images shown in D-G, and of comparable images presented throughout the manuscript. (D-G) Expression of early spleen marker genes in transverse sections through the dorsal pancreatic bud in E10.5 embryos. In situ hybridisation showing expression of Hox11 (D) and Nkx2.5 (E) (seen as red signal) in combination with pancreatic marker anti-PDX1 antibody (green). The double label highlights the relationship of spleen and pancreas location in the early mesenchyme. Expression of Wt1 (F) and capsulin/Pod1 (G) (single label shown as white signal) overlaps Hox11 expression domain and confirms that the dorsal mesenchyme is the region of the spleen rudiment. There is close association with the thickened mesodermal layer that does not express the spleen markers at a high level. The approximate position of the embryonic midline is indicated by the broken line in D. The thickened epithelium that surrounds the mesenchyme is outline by the broken lines in D and E. br, branchial arches; dp, dorsal pancreatic bud; e, eye; fb, forebrain; fl, forelimb; h, heart; hl, hindlimb; st, stomach. Scale bars: 150 µm.

 


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  		Fig. 2. SMP formation in the developing mouse embryo. (A-C) Timecourse showing the development of the SMP during mid-gestation. (A) Transverse section through an E9.5 wild-type embryo stained with antibodies for phalloidin (green) and PDX1 (red). The position of the dorsal pancreatic bud is shown with the PDX1 antibody. The white box indicates the region that is magnified (4x) in B, which highlights the organised cellular structure of the SMP that is revealed following staining for f-actin. The cells are elongated and situated perpendicular to the dorsoventral embryonic axis. (C) At E10, the SMP (white arrowheads) and spleno-pancreatic mesenchyme have grown laterally to the left. The SMP that was situated on the right side is now absent and is replaced by a thin mesothelial layer (yellow arrowheads). (D) Dh/Dh mutant embryos at E9.5 differ from wild type in that an unorganised mesenchymal layer of cells surround the midline-positioned dorsal pancreatic bud. The white box indicates the region magnified (4x) in E; the cells are round and densely packed. (F-N) At E9.5, the initially bilateral SMP is characterised by elongated nuclei (F-H). These are lost on the right-hand side in a ventral-to-dorsal direction over the course of 24 hours, and in their place nuclei resembling those in the underlying mesenchyme and adjacent mesothelia are observed (I-N). The yellow boxes in F,I and L indicate the regions magnified (4x) in G,H,J,K,M,N. Dorsal and ventral (in C only) pancreatic buds are shown in red (PDX1 antibody stain). dp, dorsal pancreatic bud. Scale bars: 60 µm in A,C-E.

 


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  		Fig. 6. Rate of cellular proliferation is high in the SMP. (A) Bright-field illumination of an E10.5 embryonic gut showing the mesenchyme that surrounds the endoderm and the refractive properties of the SMP (broken lines). (B) The gut endoderm present in A is highlighted using an antibody specific to the endodermal-specific marker HNF3ß. (D-F) Representative sections through the spleno-pancreatic region (E), the stomach region (D) and the posterior gut region (F) at positions indicated in B. Analysis of BrdU incorporation in the developing embryonic gut; cellular incorporation of BrdU is indicated by green (low level) and yellow (high level) stains. Percent incorporation of BrdU from these regions is quantified in G; the high incorporation in the SMP is highlighted in red. (C) Transverse section through the region of dorsal pancreatic bud to give an overview of BrdU incorporation in tissues. There is high incorporation in the SMP (white arrowheads) relative to other tissues. (D) The thin mesothelial layer (white arrowheads) associated with the stomach (ML in G) and the lateral coelomic mesothelium (blue arrowheads) (lateral in G), and (E) the SMP (white arrowheads) and lateral coelomic mesothelium (blue arrowheads) (lateral in G) were also analysed. Sections through the gut posterior to the pancreatic bud (F) were also analysed but are not included in G. d, duodenum; da, doral aorta; dp dorsal pancreas; flb, forelimb bud; lb, lung buds; nt, neural tube; st, stomach; vp, ventral pancreatic bud.

 


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  		Fig. 3. The SMP is under the genetic control of LR signalling. Asymmetric expression of Pitx2 (A) and Barx1 (B) on the left-hand side of the mesenchyme and associated SMP (arrowheads) is detected at E9.5. At E10.5, Pitx2 is not detected, while Barx1 (arrowheads in C) is detected in the extreme lateral domain of the spleno-pancreatic mesenchyme. Bapx1 expression is bilateral at E9.5 (D), but is higher on the leftward side (arrowheads) and is expressed in the left lateral domain at E10.5 (E). The pancreatic bud is outlined by a broken red line. (F,G) Immunohistochemical analysis of the inv mutant mouse using phalloidin (green) and PDX1 (red) antibody at E10.5. SMP formation is associated with the leftward-oriented spleno-pancreatic mesenchyme in wild type (F) and inv heterozygous embryos (data not shown), but develops in the opposite orientation in the homozygous mutant embryos (G).

 


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  		Fig. 4. Genetic control of SMP formation. (A-C) Analysis of SMP formation in the asplenic mutants Dh and Bapx1–/–, using PDX1 antibody (red) and phalloidin (green) at E10. (A) Wild-type embryos show the characteristic triangular shape of the SMP and spleno-pancreatic mesenchyme, whereas in Bapx1–/– embryos (B), although the mesenchyme grows to the left, the triangular shape is compromised and the pancreatic bud remains near the embryonic midline (broken line). In the Dh/Dh embryos (C), the SMP is lacking and no obvious leftward growth is detected. At E10.5 in Bapx1–/– embryos (E), lateral growth occurs but to a lesser extent than in wild-type embryos (D), and the pancreas remains at the midline. (F,G) Analysis of SMP formation in Fgf10–/– embryos following staining for PDX1 (red), and with propidium iodide (green). Development of the pancreatic endoderm is severely compromised in the Fgf10–/– embryos, as revealed by an apparent loss of PDX1 staining in the mutant embryos (G). Despite this, leftward growth of the spleno-pancreatic mesenchyme and SMP occurs normally (G).

 


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  		Fig. 5. Analysis of BrdU incorporation in the developing SMP. (A-C) At E9.5, BrdU incorporation is comparable on both sides of the bilateral SMP (A), with proliferating cells evenly distributed along the DV axis. This pattern is reiterated at E9.75 in sections cut through the posterior stomach (B). The initial outgrowth of the left-sided SMP in the region of the pancreatic bud is accompanied by a clustering of BrdU-positive nuclei at the prospective apex (yellow arrowheads in C). dp, dorsal pancreatic bud; st, posterior stomach.

 


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  		Fig. 7. Location of the pancreas is misplaced in the Bapx1 and Dh mutants. Three-dimensional examination of the region around the stomach at E13.5 using OPT. The endoderm is highlighted using an antibody to E-cadherin. The position of the pancreas is indicated with a yellow arrow. The wild-type embryonic gut shows the pancreas growing along an axis perpendicular to the duodenum (A). In both Dh/Dh (B) and Bapx1–/– (C) embryos, the pancreas grows along the same axis as the stomach.

 


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  		Fig. 8. Expression of FGFs in the SMP at E10.5. (A,B) Both Fgf9 and Fgf10 are expressed in the SMP and underlying mesenchyme. Fgf10 is expressed in the ventral domain of the SMP and to a lesser extent in the mesenchyme between the dorsal pancreas and the SMP (A). Fgf9 (B), and its high affinity receptor Fgfr3 (C), are expressed in the dorsal region of the SMP and towards the tip of this structure as it develops in a leftward direction. In the Bapx1–/– mutant (D), Fgf10 is downregulated in the ventral SMP, but is still detectable in the underlying mesenchyme.

 


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  		Fig. 9. Model for asymmetry in the spleno-pancreatic region. The model describes the major events that occur in lateral organ morphogenesis. The left SMP is under the influence of the LR genetic cascade and is maintained whereas the right SMP is lost. Cell proliferation in the SMP appears to be the motive force in the lateral growth that occurs in the SMP between E9.5 and E10.5 of development. During this period, the population of mesenchymal cells underlying the SMP expands considerably and the dorsal region is induced (perhaps by the associated SMP) to become spleen. Outgrowth of the spleno-pancreatic region is compromised by loss of Bapx1, as is Fgf10 expression in the ventral SMP. The high expression of Fgf10 from the SMP may be the chemotactic factor to which the dorsal pancreas responds.

 

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© The Company of Biologists Ltd 2004