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First published online 27 August 2003
doi: 10.1242/dev.00686

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Carboxypeptidase Z (CPZ) modulates Wnt signaling and regulates the development of skeletal elements in the chicken

Carsten Moeller1, Eric C. Swindell1,2, Andreas Kispert3 and Gregor Eichele1,*

1 Max Planck Institute of Experimental Endocrinology, Hannover, Germany 30625
2 Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, USA
3 Institute of Molecular Biology, Medical School Hannover, Hannover, Germany 30625



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  		Fig. 6. Evidence for binding of Wnt4 to the CRD of CPZ. (A) Schematic illustration of the constructs tested in co-immunoprecipitation assays. The domains of the proteins are indicated (for abbreviations see below). The CPZ constructs bearing the point mutation of Glu477 to Gln477 (CPZmyc/E477Q and CPZ{Delta}CRD/myc/E477Q) are not depicted. (B) Western blots of lysates of cells co-transfected with Wnt4HA and the constructs depicted in A detected with an anti-myc antibody. All proteins were expressed and had the expected molecular mass (see A). (C) A western blot of proteins of the lysates in B after co-precipitation with Wnt4HA, detected with an anti-myc antibody. sFRP-2myc (positive control), CPZmyc, CPZmyc/E477Q and CPZ{Delta}CP/myc co-precipitated with Wnt4HA. In contrast, the CRD deletion mutants, CPZ{Delta}CRD/myc and CPZ{Delta}CRD/myc/E477Q, did not bind to Wnt4HA. Note that the two bands at 25 kDa and 50 kDa in C are the immunoglobulin heavy and light chain of the primary antibody, which are also detected by the secondary antibody which is derived from the same species as the primary antibody. Since the sizes of these bands differ from those of the tested proteins, they do not interfere with detection of these proteins, but serve as a loading control. CP, carboxypeptidase domain; CRD, cysteine-rich-domain; Ni, netrin-like domain; SP, signal peptide.

 


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  		Fig. 1. Comparison of the predicted amino acid sequence of chicken CPZ with its putative human and rat orthologs. A dashed line marks the cysteine-rich-domain (CRD) in which bold and underlined residues indicate conserved cysteines, and a solid line indicates the carboxypeptidase domain. Catalytic important residues in this domain are marked in bold and underlined. An arrow indicates Glu469, the residue required for catalytic activity of cCPZ.

 


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  		Fig. 2. Expression pattern of cCPZ. (A) A HH stage 9 embryo. cCPZ transcripts are detectable in all 9 pairs of somites. (B) A HH stage 14 embryo shows expression of cCPZ in all 22 somites. Highest expression is seen in somite stage V to X [see Gossler and Hrabe de Angelis (Gossler and Hrabe de Angelis, 1998Go)] (the first somite is indicated). (C) HH stage 18 embryo showing CPZ expression in somites and apical ectodermal ridge. (D) A transverse section through a HH stage 8 embryo at the level of the second pair of somites. cCPZ is expressed throughout the entire epithelial somite. (E) A transverse section through the head of a HH stage 15 embryo at the level of the otic placode. cCPZ is expressed in paraxial head mesoderm that surrounds the notochord. (F) Section through a HH stage 15 embryo at the interlimb level. Note cCPZ expression is restricted to the sclerotome. (G) Section through a leg bud of a HH stage 20 embryo. cCPZ is strongly expressed in the AER. (H) Transverse section through a HH stage 20 embryo at the tailbud level. The entire epithelial somite expresses cCPZ. (I) cCPZ is not regulated by signals from the neural tube and/or notochord, because this gene is expressed in somites derived from segmental plate that had been separated from midline structures by HH stage 10. AER, apical ectodermal ridge; dm, dermomyotome; mc, myelencephalon; nc, notochord; np, neural plate; nt, neural tube; op, otic placode; rda, right dorsal aorta; sc, sclerotome; so, somite.

 


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  		Fig. 3. Overexpression of CPZ in somites induces Pax3. (A) Whole-mount in situ hybridization with a cCPZ probe reveals strong ectopic cCPZ expression in somites 16-19 on the injected side of the embryo. (B) Absence of ectopic expression of cCPZ on the uninjected side of the same embryo. (C) Transverse section at level of red line in A. Overexpression (blue) of cCPZ is restricted to the dermomyotome. (D) Ectopic expression of Pax3 in the hypaxial dermomyotome (arrow). (E) On the uninjected side of the same embryo as in D no ectopic Pax3 expression is seen. (F) Transverse section at level of red line in D. Note ectopic Pax3 expression in hypaxial dermomyotome (arrow); also note native epaxial Pax3 expression which is not altered by cCPZ overexpression (arrowhead). Embryos are at HH stage 21 at which time the endogenous expression of cCPZ has already declined at the axial levels depicted. Brackets in C and F delineate the subdivisions of the dermomyotome. cdm, central dermomyotome; edm, epaxial dermomyotome; hdm, hypaxial dermomyotome; lpm, lateral plate mesoderm; nt, neural tube; dm, dermomyotome; sc, sclerotome; so, somite.

 


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  		Fig. 4. Overexpression of CPZ in somites leads to scapular and rib defects on the injected side of the embryo. (A) Dorsal view of a 10-day old embryo injected with RCAS-cCPZ stained and cleared for visualizing skeletal structures. The scapula blade and the first three ribs of the embryo are severely truncated on the injected right side. (B) Line drawing of the skeletal preparation in A. (C) Injected side of a 5.5-day old embryo. Pax1 expression is absent in a region that normally contains the developing scapula (dashed outline). (D) Untreated control side of the same embryo shows a Pax1-expressing stripe of mesenchymal cells (arrow) that will give rise to the blade of the scapula. sb, scapula blade; sh, scapula head (acromium); r1, 2, 3, first, second and third rib; vc, vertebral column.

 


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  		Fig. 5. CPZ-expressing cells promote Cdx1 induction by Wnt4. (A) Cell lysates of five representative stably transfected HEK-293 cell clones express similar amounts of CPZ protein of the appropriate size (~75 kDa). The protein is found in extracts of the ECM. (B) Induction of Cdx1 in ES cell co-cultured with different mixtures of Wnt4- and CPZ-producing cells (for experimental details see Materials and methods and Results). The matrix below the diagram indicates the components present in the cocultures. ECM deposited on the culture dish was generated by untransfected HEK-293 (-) or by cells expressing wild type (CPZ) or mutant (CPZE477Q) protein. Subsequent co-culture used NIH-3T3 cells that produced either ß-gal (LacZ) (negative control) or Wnt4 together with HEK-293 cells transfected with wild-type (CPZ), mutant (CPZE477Q) CPZ protein or untransfected HEK-293 cells (-). Error bars represent standard deviation, P values smaller than 0.05 (bars 4 and 7) are indicated by one asterisk, P values smaller than 0.01 by two asterisks (bars 4 and 5) (Student's t-test).

 

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© The Company of Biologists Ltd 2003