Worm Breeder's Gazette 16(2): 36
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||International Institute of Genetics and Biophysics, Naples, ITALY|
|2||University of Calgary, Calgary, Alberta, CANADA|
Most of the Persico laboratory works on the molecular and functional characterization of two growth factors in mice: an angiogenic factor called Plgf and an EGF-like factor called Cripto. Further properties of these two factors will be described below. For the purposes of the present abstract, we are interested in the following questions: (i) are there related molecules (possibly even "homologs") in C. elegans.; (ii) what do these factors do in worms; (iii) would mammalian factors work in worms, and; (iv) would worm factors work in mammals, or at least in mammalian cells?
PLGF (Placenta derived growth factor) is a member of a family of structurally related angiogenic growth factors, all containing the 8 cysteine residues of the Platelet derived growth factor (PDGF) motif. The family includes VEGF, VEGF-B,VEGF-C and VEGF-D. Knock-out mice are viable but show subtle vascular phenotypes as adults (Carmeliet and Persico, unpublished). Plgf also has chemotactic activity on non-endothelial cells. A search of the C. elegans genomic sequence turned up one possible candidate homolog on YAC Y119D3.663; a number of cDNAs had been identified and the longest of such cDNA clones (yk305g9) was kindly provided by Yuji Kohara. The 297-aminoacid sequence derived from the full size cDNA differs from the Genefinder prediction and shows 33% identity and 46% simlarity with mouse VEGF-C, 24% identity and 40% similarity with mouse VEGF-B and 27% identity and 47% simlarity with Xenopus PDGF-B. Alignments centre on the conserved cysteine residues and are shown below. Note that the spacing of the 8 cysteine residues are the main feature conserved even between closely related mammalian species (1). A reasonable alignment can be continued C-terminal to the conserved 8-cysteine region and suggests that this particular C. elegans molecule can be aligned best with members of the family that bind to heparin.
* # # * * # ** * Ce-Ve DIKQGNDTCNLQSVC VPIPQLSDD---PQV LMYPKCYEVKQCVGS CCNS-VETCHPGTIN Vegf-C DNEWRKTQCMPREVC IDVGKEFGA--ATNT FFKPPCVSVYRCGG- CCNSEGLQCMNTSTG Vegf-B IDVYARATCQPREVV VPLSMELMG--NVVK QLVPSCVTVQRCGG- CCPDDGLECVPTGQH XLPDGFA VEEAVPAICKTRTVI YEIPRSQIDPTSANF LIWPPCVEVKRCTG- CCNTSSVKCQPSRIH # * * § Ce-Ve LVKKHVAELLYIGNG RFMFNMTKEITMEEH TSCSCFDCGSNTPQ- CAPGFVVGRSCTCEC Vegf-C YLSKTLFEITVPLSQ GPK---PVTISFANH TSCRCMSKLDVYRQV HSIIRRSLPATLPQC Vegf-B QVRMQILMIQYPSSQ LG------EMSLEEH SQCECRPKKKES--- -AVKPDSPRILCPPC XLPDGFA HRSVKVAKVEYVRKK PKLK--EVLVRLEEH LECTCTANSNSDYR- -EEETGRTRETGKKR § § § § Ce-Ve ANKEERNNCVGNATW NAETCKCECDL---- --------------K CEEGKILHKDRCDCV Vegf-C QAAN--KTCPTNYVW NNYMCRCLAQQDFIF YSNVEDDSTNGFHDV CGPNKELDEDTCQCV Vegf-B TQR---RQRPD---- -PRTCRCRCRR---- ---RR------FLHC QGRGLELNPDTCRCR XLPDGFA KRK---KLKPT---- --------------- --------------- ---------------
Note that * indicates the 8 cys residues of the PDGF motif; # indicates a.a. identity;
§ indicates Cys residues of the heparin binding protein.
To determine where the gene was expressed in worms, ~ 1.8 kb of upstream region was fused near the predicted ATG codon to GFP and lacZ expressing vectors from the Fire collection. In the one stable transgenic line examined so far (as well as in several independently produced F1 animals), expression appears confined to a subset of body wall muscle cells in late stage larvae and in adults. Our first attempts to identify these cells suggest that they do not represent a simple sub-lineage, emanating from a founder cell, for example, or from a founder cell daughter. Attempts at RNAi using the entire cDNA clone have not yet produced a strong or easily interpreted phenotype. We will continue in our attempts to define the expression pattern and RNAi phenotype. However, before proceeding to more time consuming approaches such as antibody and mutant production, we plan to produce the predicted C. elegans polypeptide in mammalian cells and then to determine if it has angiogenic activity in, for example; the chick chorioallantoic membrane assay. At this point, we have no idea how the present factor relates to the candidate VEGF receptors described by Popovici et al., in the last WBG.
Cripto is the original member of the EGF-CFC family of extracellular growth factors essential for vertebrate development: other members of this family include Zebrafish one-eyed pinhead, mouse cryptic and frog FRL1. A knock out of Cripto causes major disruptions in gastrulation, primarily affecting migration of cells through the primitive streak (2). There are a number of potential C. elegans Cripto related genes in the genomic sequences but none that is obviously the best candidate for a homolog. Thus, more for curiousity than for any other motive, we fused Cripto cDNA (including signal peptide) to a C. elegans heat shock promoter (pPD49.83), produced transgenic lines, integrated four such lines and then heat shocked embryos for 30 min at 34 degrees at a stage when the embryos have roughly 20 to 50 cells. The phenotype produced after overnight incubation is rather striking: embryos die as partially elongated and grossly malformed; in some cases they hatch but arrest. The overall appearance is lumpy, dumpy and even bumpy, with a prominent pseudo-coelomic cavity and rounded cells, possibly muscle progenitors, loosely attached to the cavity walls. At first glance, there are some superficial similarities to the phenotype produced by let-23 null mutants and one possible model is that Cripto is acting as some kind of dominant negative ligand for let-23. However, heat shocking larval stages does not produce any obvious vulval derangements (or other gross defects). It is difficult to know what should be the proper control for such an experiment; (perhaps BSA). However, we can't help noticing that the produced phenotypes are completely unlike (and in many ways more monstrous than) any phenotype we have previously observed by ectopic expression of transcription factors such as pha-4, end-1, elt-2 or elt-3. It remains to be seen whether this induced phenotype is interpretable in a satisfying manner or whether this approach could be used to identify candidate receptors.
1) Bussolino, F., Mantovani, A. & Persico, G. Molecular mechanisms of blood vessel formation. TIBS 22: 251-256 (1997)
2) Xu, C., Liguori, G., Adamson, E.D. & Persico, M.G. Specific arrest of cardiogenesis in cultured embryonic stem cells lacking Cripto-1. Developmental Biol. 196: 237-247 (1998)