New Insights into the Biophysics of Cellular Polarization during Embryogenesis

In this issue of the Biophysical Journal, Drocco et al. describe a new approach to studying the biophysics of membrane gradient polarization that is certain to provide new insights into the cellular polarization that define morphogenetic axes during embryogenesis. In a seminal 1970 Nature article (1xDiffusion in embryogenesis. Crick, F. Nature. 1970; 225: 420–422Crossref | PubMed | Scopus (405)See all References1), and even before a clear quantitative basis of molecular diffusion was achieved, Francis Crick conjectured that “a simple order-of-magnitude calculation suggests that diffusion may be the underlying mechanism in establishing morphogenetic gradients in embryonic development”. Crick based his work on the concept previously proposed by C. M. Child (2xPatterns and Problems of Development. Child, C.M. CrossrefSee all References2) and L. Wolpert (3xPositional information and the spatial pattern of cellular differentiation. Wolpert, L. J. Theor. Biol. 1969; 25: 1–47Crossref | PubMedSee all References3) that molecular morphogen gradients define axes of embryonic development. Crick recognized that if you had a site of localized morphogen insertion, that the slow diffusion coefficients of these molecules would, because of the large size of these cells, maintain a molecular gradient for extended periods of time, times sufficient to play a role in embryogenesis.Crick's principal interest was in multicellular gradients. However, in the late 1970s and early 1980s, it became clear that single cells in early embryogenesis establish molecular gradients along axes that define important cleavage planes (4xChanges in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo. Ducibella, T., Ukena, T...., and Anderson, E. J. Cell Biol. 1977; 74: 153–167Crossref | PubMedSee all References, 5xDistribution of antibody- and lectin-binding sites on dissociated blastomeres from mouse morulae: evidence for polarization at compaction. Handyside, A.H. J. Embryol. Exp. Morphol. 1980; 60: 99–116PubMedSee all References, 6xCytoplasmic polarity develops at compaction in rat and mouse embryos. Reeve, W.J.D. J. Embryol. Exp. Morphol. 1981; 62: 351–367PubMedSee all References, 7xMembrane events associated with the generation of a blastocyst. Johnson, M.H. Int. Rev. Cytol. Suppl. 1981; 12: 1–37PubMedSee all References). In the mouse, a radial axis is established during compaction at the 8-cell stage and manifests itself in the establishment of gradients in both membrane and cytoplasmic components. Ziomek and Johnson (8xCell surface interaction induces polarization of mouse 8-cell blastomeres at compaction. Ziomek, C.A. and Johnson, M.H. Cell. 1980; 21: 935–942Abstract | Full Text PDF | PubMedSee all References8) demonstrated that cell-cell contact between 8-cell murine blastomeres led to polarization of concanavalin A surface receptors, with subsequent cleavage 8-cell–16-cell occurring perpendicular to the axis defined by the gradient. This cleavage defines the earliest cellular differentiation into two cell types within the subsequent blastocyst: the inner cell mass, which becomes the embryo, and the trophectoderm, which becomes the amniotic sack and placenta.Handyside et al. (9xPolarized distribution of membrane components in two-cell mouse embryos. Handyside, A.H., Edidin, M., and Wolf, D.E. Rouxs Arch. Dev. Biol. 1987; 196: 273–278Crossref | Scopus (3)See all References9) observed a similar polarization of antigens, lectin receptors, and lipids opposite to the previous cleavage furrow at the 2-cell stage. Polarization appeared during the first 4–5 h after first cleavage and diminished, but did not disappear over the remaining period before second cleavage. This diminution was consistent with what would be predicted based upon fluorescence recovery after photobleaching measurements of membrane component lateral diffusion (10xChanges in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: indications from the lateral diffusion rates of fluorescent lipid analogs. Wolf, D.E., Edidin, M.E., and Handyside, A.H. Dev. Biol. 1981; 85: 195–198Crossref | PubMed | Scopus (19)See all References10). Thus localized morphogen insertion, coupled with known diffusion rates, were sufficient to support morphogenetic gradients until next cleavage.In their study, Handyside et al. (9xPolarized distribution of membrane components in two-cell mouse embryos. Handyside, A.H., Edidin, M., and Wolf, D.E. Rouxs Arch. Dev. Biol. 1987; 196: 273–278Crossref | Scopus (3)See all References9) assumed that newly inserted membrane material persisted over the entire cleavage-to-cleavage interval. However, more generally, if one assumes localized insertion of new membrane, the persistence of morphogenetic gradients, as well as the degree of polarity and gradient steepness, will depend upon the interplay of several factors: 1), diffusion coefficient, 2), the presence or absence of a corresponding sink at the other pole of the cell, 3), the lifetime of the morphogen at the cell surface, and 4), the rate of synthesis and insertion of new morphogen.In this issue, on pages 1807–1815, Drocco et al. introduce a new and powerful tool for assessing the effects of protein lifetime on morphogenic gradients. Coupled with imaging techniques, such as confocal microscopy, their approach enables a much more complete mathematical treatment, and therefore the ability to distinguish between models of the gradient problem, which differ in the relative effects of the four different factors discussed above. Drocco et al. have generated a fusion between the Drosophila embryo morphogen Bicoid (Bcd) (11xA gradient of bicoid protein in Drosophila embryos. Driever, W. and Nusslein-Volhard, C. Cell. 1988; 54: 83–93Abstract | Full Text PDF | PubMed | Scopus (555)See all References, 12xThe bicoid protein determines position in vthe Drosophila embryo in a concentration-dependent manner. Driever, W. and Nusslein-Volhard, C. Cell. 1988; 54: 94–105See all References) and the photoswitchable protein Dronpa (13xReversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Habuchi, S.R., Ando, R...., and Hofkens, J. Proc. Natl. Acad. Sci. USA. 2005; 102: 9511–9516Crossref | PubMed | Scopus (276)See all References13). Dronpa can be switched from its bright state into its dark state using a 496 nm Argon laser and then reconverted back to the bright state using a 405 nm diode laser. Photoconversion between the dark and light states of Dronpa, effectively allows tagging the protein present at a given point in the cell cycle and then following its subsequent evolution as well as the presence or absence of newly inserted material. They have found that Bcd lifetime changes from 50 min before the 14th mitotic cycle to only 15 min during cellularization. In addition, photoswitching of the Dronpa-Bcd fusion enables systematic mimicking of increased Bcd degradation, enabling experimental determination of the effect of morphogen lifetime on gradient shape and persistence.Because it is based on standard and widely available fusion and photoswitching techniques, the method of Drocco et al. is broadly applicable to the study of morphogens and morphogenetic gradients in a multitude of embryonic models. The method coupled with fluorescence recovery after photobleaching or fluorescence correlation spectroscopy measurements of morphogen diffusion coefficients enable a clear and definitive answer, in both biophysical and biochemical terms, to the problem of morphogenetic gradients first posed by Frances Crick (1xDiffusion in embryogenesis. Crick, F. Nature. 1970; 225: 420–422Crossref | PubMed | Scopus (405)See all References1) more than 40 years ago.