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First published online 18 April 2007
doi: 10.1242/dev.004234

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Tailbud-derived mesenchyme promotes urinary tract segmentation via BMP4 signaling

¹ Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021, USA.
² Department of Genetics and Development, Columbia University Medical Center New York, NY 10032, USA.
³ Department of Internal Medicine and Division of Basic Science, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Ronald O. Perelman Department of Dermatology, Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

View larger version (35K): [in this window] [in a new window]   Fig. 1. The renal collecting system and ureter derive from the ureteric bud. (A) Schematic of mature urinary tract with arrowhead marking the junction between the kidney and conduit system. (B) The proximal ureteric bud (UB) gives rise to the collecting tubules, whereas the distal UB gives rise to the ureters. (C,D) Two models describing the process of UB sub-division into the ureters and renal collecting system. Cts, collecting tubules; np, nephron progenitor; m, periureteral mesenchyme.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Intermediate mesoderm gives rise to the collecting system and nephron epithelia. (A) Model of the developing avian urinary tract showing the position of the gonads (g), mesonephros (ms), nephric duct (nd), metanephros (mt), ureter (u) and cloaca (c). (B,C) The ureter and intra-renal collecting system derive from rostral intermediate mesoderm. Images of representative chick embryos with replication-defective retrovirus encoding lacZ injected into the intermediate mesoderm between the axial levels of somites 6-10. Injection performed at HH st10. After 6 days (B), blue lacZ-expressing lineage-tagged cells are present in short tubules (*) extending into the mesonephros (ms), the nephric duct (nd) and the ureteric bud (ub), which has yet to undergo branching morphogenesis. Lineage-tagged cells are not present in the cloaca (c), the tissue that gives rise to the terminal portions of the conduit system. After 14 days (C) ß-gal-positive, lineage-tagged cells are present in the nephric duct (nd), the ureter (u) and the branched, metanephric (mt) intra-renal collecting system (ct). (D) Metanephric nephron epithelia derive from caudal intermediate mesoderm as determined by lacZ transfer into this tissue at HH st14. Examination of representative urinary tract 14 days after lacZ transfer caudal to somite 27 demonstrates that large populations of lineage-tagged cells are present in the metanephros (mt). Few, if any, tagged cells were present around the ureter (u) and cloaca (c). Paraffin sections of the metanephros (D, inset) demonstrate that lineage-tagged cells exhibit the morphological properties of podocytes (gp), the specialized nephron epithelia surrounding the glomerular capillary tuft, and epithelia of the tubular nephron segments (t).

View larger version (106K): [in this window] [in a new window]   Fig. 3. Tailbud-derived mesenchyme associates with the developing conduit system. (A) Whole-mount image of HH st14 embryo immediately after DiI injection into the tailbud (arrowhead). (B) The same embryo after 48 hours of incubation, including a schematic of the urinary tract. Cells deriving from the tailbud (t) are localized to the sacral aspect of the embryo and the tail. Few, if any, lineage-tagged cells were observed rostral to the hindlimb (hl). (C) Details of the schematic. At this stage, the mesonephros (ms) has formed and the nephric duct (nd) is integrated into the cloaca. The UB has not yet branched and its proximal tip is surrounded by nephron progenitors (np). Locations of transverse sections in D-F are noted. (D,E) Tailbud-derived mesenchyme associates with the distal UB as revealed by staining for Pax2 (green). The proximal UB tip (pub in D) can be identified by Pax2 expression (green), its epithelial structure and its association with Pax2-expressing nephron progenitors. Few DiItagged cells are present around the proximal UB tip. The nephric duct, which also expresses Pax2, is also visible (nd). The distal, tubular portion of the UB (dub in E) is associated with large populations of DiI-tagged cells (red). Few tagged cells deriving from the tailbud are observed near the proximal-most domain of the UB (pub). (F) The cloaca is also surrounded by large populations of DiI-tagged cells deriving from the tailbud. (G-I) The heritable lineage marker, lacZ, was transferred into the tailbud at HH st14-15 and embryos were incubated for an additional 10 days. In whole-mount preparations (G), ß-gal-expressing cells (blue in G) derived from the tailbud are present in sacral tissues, which include the cloaca (c) and the distal UB (dUB). (H) High-power examination of the cloaca indicates that lineage-tagged cells are spindle shaped and dispersed, characteristic of connective tissue. (I) Paraffin section of the ureter shows ß-gal-tagged cells derived from the tailbud in the ureteral connective tissue coat (cn). Cells deriving from the tailbud are not detected within the ureter epithelium (ep, I) or metanephros (data not shown), which are derived from rostral tissues.

View larger version (89K): [in this window] [in a new window]   Fig. 4 . Bmp4 mRNA is expressed in the tailbud and its derivatives. Whole-mount image (A) and transverse vibratome sections (B-D) of chick embryos processed for the detection of Pax2 (green) and Bmp4 (purple) mRNAs. At HH st14 (A), Pax2 mRNA is detected in the nephric duct (nd) and at the caudal-most aspect of the embryo. Bmp4 mRNA is abundantly expressed in the lateral plate (lp) and the tailbud (tb). By HH st21 (B), Bmp4 mRNA is abundantly expressed by mesenchyme surrounding the cloaca (c) and the lateral plate mesenchyme (lp), whereas Pax2 mRNA is expressed in the developing metanephros (boxed area). High-power images of developing metanephros (C,D) demonstrate that the UB (outlined by broken line) and nephrogenic mesenchyme (nm) surrounding the proximal UB tip express Pax2 mRNA. Bmp4 mRNA is expressed around the distal (dUB) but not the proximal UB (pUB). The pattern of Bmp4 expression in the developing chick urinary tract (C,D) is markedly similar, if not identical, to the pattern of Bmp4 expression in the developing murine urinary tract (E,F). Whole-mount image (E) of the urinary tract isolated from an E11.5 mouse embryo demonstrates that abundant levels of Bmp4 mRNA are expressed by mesenchyme surrounding the distal UB (dUB) and the cloaca (c). Bmp4 is not detected in the nephrogenic mesenchyme surrouding the proximal UB tip (pUB). (F) Similar results are seen in sections of E11.5 mouse metanephric kidney rudiments isolated from Bmp4+/lacz mouse embryos.

View larger version (128K): [in this window] [in a new window]   Fig. 5. Ureter differentiation occurs in zones of the UB surrounded by Bmp4-expressing mesenchyme. (A) Sections of E14.5 Bmp4+/lacz embryos show abundant ß-gal staining, indicating that Bmp4 expression persists in the mesenchyme (arrow) surrounding the distal UB (dUB). ß-gal activity can also be detected in glomerular cells (g). (B,C) Immunofluorescent analysis of E15.5 frozen sections demonstrates that this zone of the UB network (red, E-cadherin staining) differentiates into the ureter, as determined by the presence of an SMA-positive connective tissue coat (B, green) and upregulated uroplakin expression (C, green). SMA-positive cells (B) are also detected around renal arteries (ra). At E12.5 (D), the E-cadherin-positive UB network lacks both UP-positive (D) and SMA-positive cells (data not shown). However, after E12.5 rudiments were cultured for 4 days (E,F) the distal-most domain of the E-cadherin-labeled UB (red) acquires a thick, SMA-positive connective tissue coat (E, green) and exhibits upregulated expression of uroplakins (F, green). (G) In situ hybridization detection of Bmp4 in cultured rudiments indicates that ureter morphogenesis occurs only in domains of the UB bounded by Bmp4-expressing mesenchyme. Bmp4 mRNA can also be detected in glomerular podocytes (g).

View larger version (86K): [in this window] [in a new window]   Fig. 6. BMP4 signaling is required for the differentiation of the distal UB into the ureter. (A) The Cre-flox system was used to conditionally remove exons 3 and 4 of the Bmp4 gene. The presence of the loxP sites flanking the Bmp4 allele and the occurrence of the recombination event were confirmed through genotyping using specific PCR primers. (B,C) In situ analysis of Bmp4 expression was performed on rudiments isolated from E12.5 embryos and cultured for 4 days. Expression of Bmp4 mRNA is detected in the Bmp4flox/flox rudiments (B), whereas no Bmp4 is detectable in Bmp4flox/flox;Cre-Esr1 mutant rudiments (C). (D-K) Metanephric rudiments from E12.5 Bmp4flox/flox or Bmp4flox/flox;Cre-Esr1 mutant embryos were cultured for 4 days with 4-OH tamoxifen. Rudiments were analyzed as whole mounts using E-cadherin expression to visualize the UB network (red) and uroplakin (green, D-G) or SMA (green, H-K) to detect ureter differentiation. Uroplakin expression can readily be detected in Bmp4flox/flox cultures (D,F), but is difficult to detect in the Bmp4flox/flox;Cre-Esr1 rudiments (E,G). Bmp4flox/flox rudiments develop an SMA-positive coat (H,J) that condenses around the distal domain of the UB. In the Bmp4flox/flox;Cre-Esr1 rudiments, only a few unorganized SMA-positive cells are detected (I,K).

View larger version (62K): [in this window] [in a new window]   Fig. 7. Ectopic BMP signaling induces ureter morphogenesis in zones of the UB normally fated to differentiate into collecting tubules. Whole-mount images of E12.5 (A-F) or 14.5 (G-J) metanephric explants cultured for 4 days in the absence (A,B,G,H) or presence (C-F,I,J) of recombinant BMP4. The UB network is labeled with antibodies directed against E-cadherin (red, A-F; blue, G-J), differentiated ureter epithelia identified by uroplakin expression (A,C,E, green; G-J, red) and the ureteral smooth muscle coat visualized by SMA expression (B,D,F,H, green). (J) Enlargement of framed area in I. In control cultures (A,B,G,H), ureter differentiation occurs only in the distal-most domain of the UB (arrowheads) as determined by upregulated uroplakin expression (A,G) and formation of a well-organized SMA-positive (B,G) coat. SMA-positive cells can also be detected between medullary collecting tubules (ml), but are not organized into a dense connective investment around the tubules in this location. Both uroplakin-positive cells (B,E,I,J) and an SMA-positive connective tissue investment can be detected in more proximal zones of the UB (pUB) network (* referring to proximal domains of UB network) when cultures are maintained with 25 ng/ml (C,D) or 100 ng/ml BMP4 (E,F,I,J).

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