XB-ART-51356Development October 1, 2015; 142 (19): 3351-61.
The small leucine-rich repeat secreted protein Asporin induces eyes in Xenopus embryos through the IGF signalling pathway.
Small leucine-rich repeat proteoglycan (SLRP) family proteins play important roles in a number of biological events. Here, we demonstrate that the SLRP family member Asporin (ASPN) plays a crucial role in the early stages of eye development in Xenopus embryos. During embryogenesis, ASPN is broadly expressed in the neuroectoderm of the embryo. Overexpression of ASPN causes the induction of ectopic eyes. By contrast, blocking ASPN function with a morpholino oligonucleotide (ASPN-MO) inhibits eye formation, indicating that ASPN is an essential factor for eye development. Detailed molecular analyses revealed that ASPN interacts with insulin growth factor receptor (IGFR) and is essential for activating the IGF receptor-mediated intracellular signalling pathway. Moreover, ASPN perturbed the Wnt, BMP and Activin signalling pathways, suggesting that ASPN thereby creates a favourable environment in which the IGF signal can dominate. ASPN is thus a novel secreted molecule essential for eye induction through the coordination of multiple signalling pathways.
PubMed ID: 26443635
Article link: Development
Genes referenced: aspn babam2 bmpr1a egr2 en2 foxg1 igf2 ins mapk1 myc otx2 pax6 rax six6 tbxt
Morpholinos: aspn MO1 aspn MO2 aspn MO3
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|Fig. 1. Structure and expression of ASPN. (A) A phylogenic tree of SLRPs, which are divided into classes I-V. (B) Comparison of the amino acid sequences of ASPN in different species. The amino-terminal aspartic acid-rich domain and leucine-rich domains are circled with red and light blue rectangles, respectively. Asterisks mark isoleucine, leucine and valine; black circles mark cysteine (in the cysteine-rich domain); I-VIII indicate leucine-rich repeats, as predicted by a database search using LRR finder (http://www.lrrfinder.com). (C) Semiquantitative RT-PCR of ASPN and Histone4. Whole embryos from various stages were analysed by RT-PCR. uf, unfertilised eggs. (D-G) Spatial expression of ASPN in Xenopus embryos at neurula stage (st18; anterior view; D) compared with Pax6 expression at neurula stage (st18; anterior view; E) and ASPN expression in early tailbud (st22; lateral view; F) and tadpole (st35; G) stages was analysed by in situ hybridisation. The neural plate border (D) and the presumptive eye region (F) are indicated by yellow arrowheads. (H) Expression levels of ASPN in various types of explants, as assayed by qRT-PCR. Animal caps [control (i) or injected with mRNAs of Chd (ii) and Chd+Wnt8 (iii)] and dorsal marginal zone (DMZ; iv) and ventral marginal zone (VMZ; v) were prepared at stage 10.5 and assayed at stage 18. Error bars represent s.e.m.|
|Fig. 2. ASPN induces eye-like structures. (A,B) Arrows indicate ectopic eye-like structures that appeared following overexpression of ASPN mRNA (B). (C-H) Injection of ASPN mRNA induces an eye-like structure. In contrast to the embryos injected with 3 ng of control (β-Galactosidase) mRNA (C-C′,F), either 1 ng (D-D′,G) or 3 ng (E-E′,H) of ASPN mRNA at the 4-cell stage induced ectopic pigmented structures (arrows) at stage 42. The whole structure was imaged (C-E′), or Haematoxylin and Eosin staining was performed with sectioned samples (F-H). (I) Quantification of the phenotypes induced by injection of ASPN mRNA at various concentrations. The phenotypes were divided into four categories: embryos with normal eyes, with enlarged eyes (as in D-D′), with ectopic eyes (as in E-E′) and with a short axis. (J-O) Immunohistochemistry performed on sections of the pigmented eye-like structure induced by ASPN show that it contains eye-specific components. Embryonic eyes (J,L,N) and the pigmented structure induced by injection of ASPN (K,M,O) were analysed at stage 42 with β-Crystallin (J,K), Glutamine Synthetase (L,M) and Hu-C/Hu-D (N,O) antibodies. Green, immunohistochemical signal; blue, DAPI. (P) The phenotypes found following injection of SLRP family members. SLRP family members were injected at 3 ng into a dorsal animal blastomere at the 4-cell stage and the phenotypes were categorised at stage 42. Key is shown in I.|
|Fig. 3. ASPN induces forebrain marker genes both in vivo and in vitro. (A-L) Forebrain marker genes were increased at the expense of posterior markers in vivo. The tracer β-Galactosidase (light blue product) was injected without (A,C,E,G,I,K) or with (B,D,F,H,J,L) ASPN mRNA and embryos were analysed by in situ hybridisation with Rx (A,B), Pax6 (C,D), Otx2 (E,F), FoxG1 (G,H), En2 (I,J) or Krox20 (K,L) probes at stage 18. Affected areas are indicated by arrowheads. (M) Control (lane 2) or ASPN-injected (lane 3) animal cap explants were analysed by semi-quantitative RT-PCR. Whole embryos (lane 1) were used as a positive control for the PCR.|
|Fig. 4. ASPN is required for eye development. (A-C) Representative images from the injection of control-MO (A), ASPNMO1 (B) and ASPN-MO1 together with the coding region of ASPN (ASPNCDR) mRNA (C). (D) Quantification of the phenotypes. For the rescue experiment, embryos were injected with either 20 ng ASPN-MO1 and 1 ng ASPNCDR, or 20 ng ASPN-MO1 and 3 ng ASPNCDR, and the phenotypes analysed at stage 41. (E-X) Expression of marker genes caused by ASPN-MO1. Either control-MO (E,G,I,K,M,O,Q,S,U,W) or ASPN-MO (F,H,J,L,N,P,R,T,V,X) was injected together with β-Galactosidase mRNA as a tracer (light blue product) and embryos were analysed at stage 17 (E,F,I,J,M,N,Q,R,U,V) or stage 22 (G,H,K,L,O,P,S,T,W,X) by in situ hybridisation with the probes of Rx (E,F), Six3 (G,H), Pax6 (I,J), Six6 (K,L), Otx2 (M-P), En2 (Q-T) and Krox20 (U-X). Arrowheads in F and J indicate affected areas. (Y) ASPN is essential for the induction of EFTFs by Chordin (Chd). Animal caps of control (i; black bars), Chd-injected (ii; blue bars) and Chd+ASPN-MO-injected (iii; red bars) embryos were prepared and the animal caps were analysed at stage 22 by qRT-PCR (*P<0.01; Student’s t-test). Error bars represent s.e.m.|
|Fig. 5. Cooperation of ASPN and IGF is essential for eye development. (A) ASPN activates ERK and AKT. Conditioned media taken from control GFP (lane 1), ASPN (lane 2) or IGF2 (lane 3) expressing cells were applied to HEK 293 cells for 20 min.Western blotting analysis was performed with antibodies for phosphorylated ERK (p-ERK), ERK, phosphorylated AKT (p-AKT) and AKT. (B) ASPN physically interacts with IGF1R. HEK 293 cells were transfected with expression vectors carrying IGF1R (lanes 1,2) and ASPN (lane 2) and co-immunoprecipitation analysis was performed with the IGF1R antibody and detected with the myc antibody. IB, immunoblotting; IP, immunoprecipitation. (C-I) Embryonic eye formation requires both ASPN and IGF signals. (C-G) Embryos were injected with 3 ng β-Galactosidase mRNA (control: C), 1 ng ASPN mRNA (D), 1 ng ASPN+3 ng dnIGFR mRNAs (E), 1 ng IGF2 mRNA+20 ng control-MO (F) or 1 ng IGF2 mRNA+10 ng ASPN-MO (G) into the dorsal blastomere at the 4-cell stage and phenotypes were evaluated at stage 42. Affected areas are indicated with yellow arrowheads. (H,I) The same combination of mRNAs and morpholinos were injected. Animal caps were prepared and analysed at stage 22 for Pax6 and Rx2a expression with qRT-PCR (*P<0.01; Student’s t-test). Error bars represent s.e.m.|
|Fig. 6. ASPN inhibits multiple signal molecules. (A) ASPN blocks endogenous Activin, BMP and Wnt signals, as examined by luciferase assays. ARE-luc, BRE-luc or TOPFLASH reporter constructs were injected with 1 ng β-Galactosidase (control), 100 pg Xnr1 mRNA (for ARE), 100 pg BMP4 mRNA (for BRE),100 pg Wnt8 mRNA (for TOPFLASH), 100 pg Xnr1+1 ng ASPN mRNAs (for ARE), 100 pg BMP4+1 ng ASPN mRNAs (for BRE) or 100 pg Wnt8+1 ng ASPN mRNAs (for TOPFLASH) and were assayed at stage 12. (B) ASPN inhibits the Nodal signalling pathway. Animal caps injected with control or ASPN mRNA were prepared at stage 9 and cultured with control medium or medium containing human Nodal protein until stage 10.5. Mix.2 expression was analysed by qRT-PCR. (C,D) Xbra expression was inhibited by ASPN, as analysed by in situ hybridisation. The β-Galactosidase mRNA (light blue product) was injected without (C) or with (D) ASPN mRNAs into one blastomere at the equator region of 4-cell stage embryos and embryos were cultured until stage 10.5. Affected areas are indicated with arrowheads. (E) ASPN has neural-inducing activity. Animal caps injected with 500 pg Chd (lane 3) or 1 ng ASPN (lane 4) mRNAs were analysed at stage 14 by semi-quantitative PCR. (F) ASPN inhibits the Wnt signalling pathway. Animal caps injected with Wnt8 and ASPN mRNAs were prepared and the expression of Xnr3 was analysed at stage 10.5. (G-I) ASPN forms complexes with BMP4 (G), Xnr1 (H) and Wnt8 (I) proteins. In order to avoid artificial interactions in the same cells, each expression construct was separately transfected into HEK293 cells and cells were combined on the following day as indicated. The cell lysates were collected after two additional days and immunoprecipitation (IP) was performed with the HA antibody and western blotting (IB) was performed with the FLAG (G) or myc (H,I) antibodies (*P<0.01; **P<0.05; Student’s t-test). Error bars represent s.e.m.|
|Figure S1. Expression of ASPN and other related genes in various explants. Expression levels of ASPN (A), Otx2 (B) and Krox20 (C) in various types of explants, as assayed by qRT-PCR. Animal caps (control (i) or injected with mRNAs of 500 pg IGF2 (ii), 500 pg Chd (iii) or 500pg Chd + 100 pg Wnt8 (iv)) and dorsal marginal zone (DMZ; v) and ventral marginal zone (VMZ; vi) were prepared at stage 10.5 and assayed at stage 18. Note that the data indicated with (†) are identical to those in Fig.1H.|
|Figure S2. Characterisation of Lumican and Decorin. (A-C) Representative images of the embryos injected with 3 ng ASPN (A), 3 ng Lumican (B) and 3 ng Decorin (C) mRNAs. (D) Differential activation of ERK and AKT by SLRP proteins. Control (i), ASPN-myc (ii), Lumican-myc (iii) or Decorin-myc (iv) expression media were prepared and applied onto HEK293 cells as in Fig. 5A.|
|Figure S3. Designation of morpholino oligonucleotides against ASPN and the phenotypes caused by the ASPN-MO2. (A) In addition to ASPNa, which this study is based on, we found another genome sequence probably due to the pseudotetraploidity, and termed it ASPNb. The nucleotide sequences (black characters) around the start codon (circled) of Xenopus ASPN and the sequences of ASPN-MO1 (red) ASPNa-MO2 (blue) and ASPNb-MO2 (purple) are shown. (B-D) Representative images from the injection of 20 ng control-MO (B), 20 ng ASPN-MO2 (C) and 20ng ASPN-MO2 together with 1 ng of the coding region of ASPN (ASPNCDR) mRNA (D). (E) Quantification of the phenotypes. For the rescue experiment embryos were injected with either 20 ng ASPN-MO2 and 1 ng ASPNCDR, or 20 ng ASPN-MO2 and 3 ng ASPNCDR and the phenotypes analysed at stage 41.|
|Figure S4. Both IGF and ASPN are required for the full activation of ERK. Animal cap explants were prepared from 3 ng control -Galactosidase (i,ii,iv), 3ng dnIGFR mRNA (iii), 20 ng control-MO (v) or 20 ng ASPN-MO (vi) injected embryos and were incubated with the conditioned media expressing control (i,iv), ASPN (ii,iii) or IGF2 (v,vi) for 20 minutes. The explants were analysed by western blotting using phosphor-ERK or ERK antibodies.|
|Figure S5 Interactions between ASPN and other molecules. The expression plasmids encoding ASPN-HA, IGF2-myc (A), Activin receptor (ActR)-FLAG (B), BMP receptor (BMPR)-FLAG (C) and Fzd4-CRD (the cysteine-rich domain in the extracellular part of Frz4)-myc-FLAG (D) were transfected as in Fig. 6G-I. The cell extracts were analysed by coimmunoprecipitation assays.|
|Figure S6. The phenotypes caused by the ventral injection of ASPN mRNA. 3 ng of ASPN mRNA was injected at the equator regions of one of the blastomeres at 4-cell stage and the phenotype observed at stage 42. In contrast to the control embryos (A), the injected embryos exhibited shortened bodies (B). Table|
|aspn (asporin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.|
|aspn (asporin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior right, dorsal up.|
|aspn (asporin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anterior right, dorsal up.|