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First published online 6 February 2008
doi: 10.1242/dev.013359

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R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis

¹ Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children's Hospital Medical Center and The University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
² Division of Developmental Biology, Cincinnati Children's Hospital Medical Center and The University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Rspo2 is disrupted in the transgene insertional mutation footless. (A) Rspo2 gene. Untranslated exons are shown in black, arrows indicate the location of PCR primers. (B) Northern blot of E11.0 embryo total RNA. Arrow indicates full-length transcript; arrowhead indicates alternatively spliced transcript. (C) RT-PCR of E11.0 embryonic cDNA. The primers for exons 3-6 spanning the transgene integration site detected only small amounts of product in Rspo2Tg/Tg embryos.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Structural requirements for activation of Wnt signaling by Rspo2. (A) Domains of Rspo2. Signal peptide (light gray) followed by a cysteine-rich domain, a furin-like domain (black box), a TSP1 domain (gray box) and a basic C-terminus. Arrowheads indicate exon junctions. (B) Western blot analysis. An anti-AP antibody detected appropriately sized Rspo2AP fusion proteins in concentrated conditioned media. (C) Conditioned media containing the indicated Rspo2AP fusion proteins was incubated with untransfected HEK293T cells and cell bound AP activity determined. (D) Binding affinity of Rspo2AP fusion products to heparin agarose, only Rspo2AP and 212-243AP possessed a high affinity for heparin. (E) HEK293T cells co-transfected with TOPFLASH and pRL-TK were exposed to the indicated conditioned media. Firefly luciferase was normalized to Renilla luciferase activity. Activity induced in response to AP conditioned media was set to 1. Furin domain-containing constructs activated TOPFLASH. (F) Flag-tagged Lrp6 expression constructs with the indicated domains deleted were co-transfected into HEK293T cells with TOPFLASH and pRL-TK. Normalized luciferase activity in pcDNA3.1-transfected cells exposed to AP conditioned media was set to 1. Bars represent the mean±s.e.m. induction. Inset shows western blot of cell lysates from parallel wells. An anti-flag antibody detected expression of appropriately sized Lrp6 proteins.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Rspo2 expression is regulated by Sp8 in the limb. Whole-mount in situ hybridization was performed on wild-type (A,B,D-H,J), Sp8-/- (C) and Rspo2Tg/Tg (K) embryos of the indicated age [embryonic day (E)]. (A-H) Rspo2 antisense riboprobe corresponding to sequences in EST AK011587. (J,K) Sp8 antisense riboprobe. (A-D) Telencephalon expression (white arrowheads). (A,B) AER precursors (red arrowheads). (B,D) Mature AER (red arrows). (B) Transient domain at base of hindlimb (black arrowhead); inset highlights proximal mesenchymal expression domain. (C) Rspo2 expression is undetectable in limb buds (black arrowheads). (D) Genital ridge expression (black arrowhead). (E) Expression in nasal pits. (F-H) Rspo2 expression in laryngeal regions (arrows) and lung mesenchyme (arrowheads). (I) Real-time RT-PCR products amplified from cDNA prepared from stage 2 hindlimb bud ectoderm from Sp8-/- (lane 1) or Sp8+/+ (lane 2), and stage 4 Sp8+/+ (lane 3) embryos. Rspo2 was dramatically decreased in Sp8-/- ectoderm and increased during limb outgrowth in control embryos, whereas the expression of other AER markers was changed less than twofold. (J,K) Arrows indicate differences in Sp8 expression in the posterior AER margin; expression is weaker in Rspo2Tg/Tg forelimb AER (arrowhead).

View larger version (46K): [in this window] [in a new window]   Fig. 4. Rspo2 and Lrp6 interact during morphogenesis of the limbs. Alcian Blue and Alizarin Red stained E18.5 right forelimbs (A-F) and left hindlimbs (G-L). Asterisks in A-D and G-J indicate absence of digits. Arrowheads in A and C indicate absence of a radius. Arrow in C indicates malformation of the humerus. Arrowhead in G-I indicate absence of the fibula. Arrow in I indicates absence of the femur.

View larger version (179K): [in this window] [in a new window]   Fig. 5. Laryngeal-tracheal-bronchial and lung malformations in Rspo2/Lrp6 mutant mice. (A-F) Alcian Blue stained cartilages in the larynx, trachea and bronchi from E18.5 fetuses of indicated genotypes. Asterisks in B and C indicate fusion of the arytenoids to the cricoid cartilage. (C) Arrow indicates the cricoid; cartilage is absent on the dorsal aspect of the ring, also observed in B and D. (F) The SftpC-rtTA transgene does not target cells within the larynx epithelium and only tracheal ring defects were observed. (G-N) Immunohistochemical analysis for pro-SftpC, a type II epithelial cell marker (G-J); and Ccsp, a nonciliated bronchiolar cell marker (K-N). (G,K) Rspo2+/+;Lrp6+/+; (H,L) Rspo2Tg/Tg;Lrp6+/+; (I,M) Rspo2Tg/Tg;Lrp6-/-; (J,N) Rspo2+/+;Lrp6-/-. Scale bars: 1 mm in F; 10 µm in J and N.

View larger version (89K): [in this window] [in a new window]   Fig. 6. Branching defects in Rspo2Tg/Tg lungs. Organ culture of E11.5 lungs in the presence of conditioned media containing either AP or Rspo2 212-243AP. (A) Rspo2 212-243AP partially rescued the branching defect observed in Rspo2Tg/Tg lung buds. (B) Counted epithelial tips of mutant lung buds in culture. (C,D) Rspo2+/+ and Rspo2+/Tg lung buds grew similarly in culture in response to either AP or Rspo2 212-243AP. (D) Number of distal tips of individual lung buds observed within a representative experiment.

View larger version (70K): [in this window] [in a new window]   Fig. 7. Rspo2Tg/Tg lungs exhibit reduced Wnt signaling. The Rspo2Tg allele was crossed into the Wnt signaling reporter TOPGAL line and β-galactosidase activity examined in whole-mount lung specimens at E11.5: Rspo2+/+ (A), Rspo2Tg/Tg (B,C,E). A, B and E are littermates. White arrows indicate high levels of Wnt signaling activity in the distal tips of wild-type lungs compared with the lower level in the distal tips of mutants (arrowheads). (D) Quantitation of β-galactosidase activity in E11.5 lung buds. (F) Semi-quantitative real-time RT-PCR of E11.5 cDNA samples, indicating that Irx3 is reduced threefold. (G,H) Sox9 is expressed in a normal proximal distal pattern in Rspo2+/+ (G) and Rspo2Tg/Tg (H) lungs.