spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online February 18, 2004
doi: 10.1242/10.1242/dev.00997

Development 131, 1187-1194 (2004)
Published by The Company of Biologists 2004
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Conover, C. A.
Right arrow Articles by van Deursen, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Conover, C. A.
Right arrow Articles by van Deursen, J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development

Cheryl A. Conover1,*, Laurie K. Bale1, Michael T. Overgaard2, Edward W. Johnstone1, Ulla H. Laursen2, Ernst-Martin Füchtbauer2, Claus Oxvig2 and Jan van Deursen3

1 The Division of Endocrinology, Metabolism and Nutrition, Endocrine Research Unit, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN 55905, USA
2 The University of Aarhus, Department of Molecular Biology, Science Park, Gustav Wieds Vej 10C, DK-8000, Aarhus C, Denmark
3 The Department of Pediatric and Adolescent Medicine, and Department of Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN 55905, USA



View larger version (37K):

[in a new window]
  		Fig. 1. Generation of PAPPA-null mice. (A) Schematic representation of the mouse gene in the region of exons 3 and 4 of the PAPPA locus, the replacement vector and the targeted allele. The position of the probe used for Southern analysis is indicated by the dark gray bar, and the sizes of the endogenous and targeted BamHI (B) genomic DNA fragments recognized by this probe are shown. An example of genotyping of mouse tail DNA is shown in the insert. Wild-type mice have a single 15 kb band (lanes 1, 8), heterozygous mice have both 15 kb and 2.6 kb bands (lanes 6, 7), and homozygous mutants have a single 2.6 kb band (lanes 2-5). (B) Weights of wild-type (+/+), heterozygous (+/-) and PAPPA-deficient (-/-) mice at birth. Results are mean±s.e.m.; n=20 for each genotype. *, significantly different from wild-type, P<0.0001. (C) Growth curves of wild-type ({diamondsuit}), heterozygous ({blacksquare}) and homozygous ({blacktriangleup}) PAPPA-/- mutant mice. Values are mean±s.e.m. of 28-141 individual weights. (D) RT-PCR for PAPPA mRNA expression (upper panel) in tissues from wild-type (WT) and PAPPA-/- mice. k, kidney; h, heart; li, liver; f, femur; t, tibia; b, brain; c, calvaria; lu, lung. -, negative control; +, positive control. Lower panel shows ethidium bromide staining of tissue 28S and 18S RNA to validate integrity and loading. (E) IGFBP4 proteolysis in medium conditioned by embryonic fibroblasts derived from wild-type (WT) and PAPPA-/- mice. 125I-IGFBP4 was incubated in MEF-conditioned medium without (-) or with (+) IGF2 for 6 hours. Reaction products were analyzed by SDS-PAGE and autoradiography. Arrows indicate intact IGFBP4 and 18 kD and 14 kD proteolytic fragments.

 


View larger version (21K):

[in a new window]
  		Fig. 5. Effect of PAPPA on IGF-mediated growth responses in mouse embryonic fibroblasts. (A) Fibroblasts from wild-type (WT) and PAPPA-/- embryos were incubated for 48 hours prior to the addition of recombinant wild-type IGFBP4 (lanes 3, 4) or protease-resistant IGFBP4 (lanes 5, 6). Lanes 1, 2 are cells with no IGFBP4 added. After an additional 1 hour of incubation, cells were lysed and immunoprecipitated with either antibody against the type I IGF receptor (even number lanes) or non-specific IgG (odd number lanes), as described in the Materials and methods. Immunoprecipitates were run on SDS-PAGE under reducing conditions and immunoblotted with phosphotyrosine antibody (Tyr-P IGF1R) or type 2GF receptor antibody (IGF1R). (B) Fibroblasts from WT (n=5) and PAPPA-/- embryos (n=8) were incubated for 48 hours prior to the addition of 25 nM IGFBP4 and 5 nM IGF (black bars) or IGF analog (gray bars). Results of experiments using IGF1 and IGF2 [and corresponding analogs, des(1-3)IGF1 and des(1-6)IGF2] were combined as they gave equivalent responses (see Table 2). Addition of 25 nM IGFBP3 and 5 nM IGF is represented by the white bars. [3H]Thymidine incorporation was measured as described in the Materials and methods. Data (mean±s.e.m.) are expressed as percent of control. *, significantly different from control, P<0.01.

 


View larger version (38K):

[in a new window]
  		Fig. 2. Skeletal development. (A) At E13.5, mineralization of the clavicle (arrows) is delayed in the PAPPA-/- compared with wild-type mice. (B) At E16.5, no caudal vertebrae were mineralized in the PAPPA-/- mouse, whereas three to four caudal vertebral elements were undergoing mineralization in wild-type littermates (asterisk). The frontal (f) and interparietal (i) bones of the cranial vault are both undergoing mineralization at E16.5, but these processes are far less complete in PAPPA-/- embryos. (C) Close-up of digits (arrow) and vertebrae (asterisk). Note an additional metatarsal had initiated mineralization in the wild-type compared with the PAPPA-/- mice (arrow). (D) Close-up of cranial vault. The nasal (n), parietal (p) and occipital (o) bones are also delayed.

 


View larger version (79K):

[in a new window]
  		Fig. 3. In situ hybridization for PAPPA expresssion in wild-type mouse embryos. At (A) E8.5 and (B) E9.5, PAPPA expression is mainly found in the developing foregut (fg) and hindgut (hg). Staining in the optic and otic vesicle is an artifact. At (C) E10.5 and (D) E11.5, PAPPA expression is seen in the cells resembling migrating neural crest cells (arrowhead), in branchial arches (asterisk), in the forelimbs (fl) and in a segmented pattern in the tail (t). At (E) E12.5, (F) E13.5 and (G) E14.5, PAPPA expression persisted in the developing limbs, tail, nasal region and the developing outer ear. Note that in F an albino embryo is shown, indicating that there is no PAPPA expression in the developing eye. (H) Higher magnification of the tail at E10.5 to demonstrate the segmented expression in the developing somites. (I) Higher magnification of the forelimb of an E13.5 embryo showing PAPPA expression in the developing joints of the digits.

 


View larger version (78K):

[in a new window]
  		Fig. 4. In situ hybridization for IGF2 and IGFBP4 expression. Wild-type (A,B,C) and PAPPA-/- (D,E,F) E12.5 embryos hybridized with digoxigenin-labeled antisense RNA probes for IGF2 (A,B,D,E) or IGFBP4 (C,F). (B,C,E,F) Dorsal views, anterior is to the upper right. Strong expression is seen lateral to the neural tube (nt), most probably in the spinal ganglia, as well as in the forelimbs (fl), hindlimbs (hl) and tail (t).

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2004