In situ analysis of embryos with receptor or ligand fusion protein probes

In situ staining of embryos with receptor or ligand fusion protein probes, initially described in 1994 [1xIdentification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases. Cheng, H.-J. and Flanagan, J.G. Cell. 1994; 79: 157–168Abstract | Full Text PDF | PubMed | Scopus (281)See all References, 2xComplementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cheng, H.-J., Nakamoto, M., Bergemann, A.D., and Flanagan, J.G. Cell. 1995; 82: 371–381Abstract | Full Text PDF | PubMedSee all References], is an increasingly widely used technique to study the ephrins and other molecular families [3xAlkaline phosphatase fusions of ligands or receptors as in situ probes for staining of cells, tissues and embryos. Flanagan, J.G., Cheng, H.J., Feldheim, D., Hattori, M., Lu, Q., and Vanderhaeghen, P. Meth Enzymol. 2000; See all References][3]. In a recent letter to Current Biology, Sobieszczuk and Wilkinson [4xMasking of Eph receptors and ephrins. Sobieszczuk, D.F. and Wilkinson, D.G. Curr Biol. 1999; 9: R469–R470Abstract | Full Text | Full Text PDF | PubMed | Scopus (26)See all References][4] describe interesting results showing that overexpression of a ligand can result in masking of its receptor, as assessed with fusion protein probes, emphasizing that the results obtained with such probes always have to be interpreted carefully. As this in situ technique is relatively new, it may be useful to consider further its advantages and limitations in comparison with other available methods.Three other techniques used to study molecular distributions in vertebrate embryos are RNA in situ hybridization [5xDetection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Wilkinson, D.G. and Nieto, M.A. Meth Enzymol. 1993; 225: 361–373CrossRef | PubMed | Scopus (598)See all References][5], immunolocalization [6xAntibodies: a laboratory manual. Harlow, E. and Lane, D. See all References][6], and reporter gene insertion [7xThe identification of new genes: gene trapping in transgenic mice. Skarnes, W.C. Curr Opin Biotech. 1993; 4: 684–689CrossRef | PubMed | Scopus (13)See all References][7]. It is always important to bear in mind that, by their nature, none of the available techniques can necessarily be assumed to give the biologically ‘accurate’ expression pattern. Each of them gives a different type of information, and each has unique advantages, as well as limitations.Briefly, RNA in situ hybridization is relatively quick and reliable, and the probes and conditions can be carefully adjusted for a low risk of cross-reactivity. However, it provides no direct information on the pattern or subcellular distribution of proteins, the functional species one is usually interested in. Reporter gene insertion by homologous knock-in or gene trapping can show the RNA expression pattern of the targeted gene and can be highly sensitive and specific. It may not necessarily reflect the natural spatial and dynamic aspects of RNA or protein expression, however. Immunolocalization can show detailed protein distributions and can be highly specific. On the other hand, here too, there are well known limitations. Raising antibodies is time-consuming, expensive, and sometimes unsuccessful. Results must be interpreted with caution, as the antibody may cross-react with multiple proteins. Furthermore, the antibody may bind to, or be affected by, post-translational modifications such as carbohydrate epitopes, and may detect degradation products and other non-functional species.Ligand or receptor probes are generally made as a fusion protein with an alkaline phosphatase tag [1xIdentification and cloning of ELF-1, a developmentally expressed ligand for the Mek4 and Sek receptor tyrosine kinases. Cheng, H.-J. and Flanagan, J.G. Cell. 1994; 79: 157–168Abstract | Full Text PDF | PubMed | Scopus (281)See all References, 2xComplementary gradients in expression and binding of ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cheng, H.-J., Nakamoto, M., Bergemann, A.D., and Flanagan, J.G. Cell. 1995; 82: 371–381Abstract | Full Text PDF | PubMedSee all References, 8xThe kit ligand: a cell surface molecule altered in Steel mutant fibroblasts. Flanagan, J.G. and Leder, P. Cell. 1990; 63: 185–194Abstract | Full Text PDF | PubMed | Scopus (380)See all References] or an immunoglobulin Fc tag [9xCD44 is the principal cell surface receptor for hyaluronate. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C.B., and Seed, B. Cell. 1990; 61: 1303–1313Abstract | Full Text PDF | PubMed | Scopus (1238)See all References, 10xEph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Gale, N.W., Holland, S.J., Valenzuela, D.M., Flenniken, A., Pan, L., Ryan, T.E., Henkemeyer, M., Strebhardt, K., Hirai, H., Wilkinson, D.G. et al. Neuron. 1996; 17: 9–19Abstract | Full Text | Full Text PDF | PubMed | Scopus (586)See all References]. They can be produced far more quickly than antibodies. In our experience, production of fusion proteins that retain binding activity has been reliable, though this will depend on the properties of the individual receptor or ligand. In situ detection procedures are quick and simple, usually taking only a few hours. The fusion probes can be used in the same types of procedure as antibodies, but differ in that they can detect natural ligand–receptor interactions. It is therefore important to recognize that the information gained is qualitatively different. One obvious advantage is that one can find novel binding partners, a feature that has been used to identify a large number of new receptors and ligands. Another unique advantage is that, in the case of receptors that bind to multiple ligands (or vice versa), one can obtain global evidence on the overall distribution of ligands for that receptor. To look at this another way, one can ask a question about the probe — what distribution of ligands can this receptor detect in the embryo? In addition, the technique is presumably selective for functional, available forms of molecules that are capable of ligand–receptor binding.As with the other techniques, though, one must be cautious in interpreting the results. It may be hard to draw conclusions about any single molecule, since the technique is liable to simultaneously detect multiple binding partners. In addition, formation of pre-existing endogenous receptor–ligand complexes may mask binding sites for the probe. For both of these reasons, it can be informative to compare the results with another technique, such as RNA hybridization.Sobieszczuk and Wilkinson [4xMasking of Eph receptors and ephrins. Sobieszczuk, D.F. and Wilkinson, D.G. Curr Biol. 1999; 9: R469–R470Abstract | Full Text | Full Text PDF | PubMed | Scopus (26)See all References][4] show that transgenic overexpression of ephrin-A5 can reduce the binding of an ephrin-A5–Fc probe to embryos, providing direct evidence that masking can indeed occur in an embryo. As they point out, it is reassuring that previous studies of ephrins and Eph receptors have generally shown a reasonably good match of fusion-protein binding, when compared with RNA hybridization. Their report is valuable, though, in emphasizing that masking is a mechanism to consider, and indicating that ephrins and their receptors may be less compartmentalized, and more overlapping, than might be concluded based on binding of receptor and ligand fusion proteins [4xMasking of Eph receptors and ephrins. Sobieszczuk, D.F. and Wilkinson, D.G. Curr Biol. 1999; 9: R469–R470Abstract | Full Text | Full Text PDF | PubMed | Scopus (26)See all References][4].Does this mean that masking can be dismissed as a misleading artifact of the technique? Not necessarily. To give one example, recent studies of retinotectal mapping, by in vitro assays, in vivo overexpression [11xModulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons. Hornberger, M.R., Dutting, D., Ciossek, T., Yamada, T., Handwerker, C., Lang, S., Weth, F., Huf, J., Wessel, R., Logan, C. et al. Neuron. 1999; 22: 731–742Abstract | Full Text | Full Text PDF | PubMed | Scopus (258)See all References][11], and gene knockout analysis (D. Feldheim and J.G.F., unpublished observations) indicate that a reduced availability of Eph receptors due to ligand co-expression may be a genuine regulatory mechanism in normal development. Viewed in this light, if fusion protein probes are selective for unmasked sites, this might actually be seen as a useful aspect of the technique that could help in assessing the distribution of available receptor or ligand.So, the question remains, which of the available techniques for expression pattern analysis most accurately reflects biological reality? The best answer may be ‘all of the above’. No one technique necessarily gives the whole picture. Each can provide qualitatively different types of information, and all can help in the quest to understand biological systems.