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• W2026671350 abstract "We highlight crucial technological progress of the past ten years that permits quantitative analysis of cellular behavior. Adapting these methods to the study of embryogenesis will be essential to advance our understanding of development in the coming decade. We highlight crucial technological progress of the past ten years that permits quantitative analysis of cellular behavior. Adapting these methods to the study of embryogenesis will be essential to advance our understanding of development in the coming decade. Cell biology can be defined as the attempt to understand how cellular behavior emerges from the combined activities of biologically interesting molecules. To do this for any molecule involved in a particular process, it is crucial to determine the cues controlling its spatiotemporal expression, the strength of its interactions with other factors, the full set of downstream consequences of its biochemical activity, and probabilities of finding it in a given state. Although tissue culture cells can provide many useful mechanistic insights, such quantitative analyses ultimately must be done in intact living organisms. This final challenge brings many cell biological problems into the purview of developmental biology, since among living organisms, embryos are by far the most abundant and the easiest to work with. The cells of a developing metazoan embryo are not only beautiful but also exhibit the full spectrum of biological functions of interest to cell biologists. Indeed, the origin of cell biology can be traced back to experimental embryology (Dawes-Hoang and Wieschaus, 2001Dawes-Hoang R.E. Wieschaus E.F. Dev. Cell. 2001; 1: 27-36Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), and developing embryos continue to represent the most suitable contexts in which to address cell biological questions of fundamental importance, especially regarding signal propagation and coordinated cell movement. Much of cell and developmental biology over the past 30 years has focused on identifying factors necessary for a given process and qualitatively describing their spatiotemporal expression and the effects of their absence or misregulation. Only in rare cases have quantitative biophysical characteristics, such as a factor's enzymatic rates or binding affinities, been related to its effects at the cellular or tissue level. Few studies have attempted to measure protein concentrations or lifetimes, despite the fact that such values must determine the magnitude of a factor's effect. In addition, even with the plethora of tools to manipulate expression and/or activity, in many cases we do not know precisely where, when, or how much of a given biological molecule is necessary to elicit the behaviors we observe at a cellular or embryonic level. In the absence of such knowledge, it is difficult or impossible to move beyond qualitative descriptions to address outstanding questions of precision, reproducibility, modularity, and evolvability in patterning and development. We believe that the next 20 years of developmental and cell biology will be an especially exciting period because recent progress in imaging and quantification will finally allow such quantitative analyses. The use of these approaches already represents a vital component of studies in cell biology, and we advocate their adaptation to developmental contexts so that quantitatively rigorous models may be devised and tested to describe how developmental processes arise from biochemical and biophysical characteristics of the underlying molecules. Gradients of secreted factors are now widely acknowledged as a predominant means of embryonic patterning. Yet, for all the attention paid to morphogen gradients, significant questions regarding their establishment, stability, and interpretation have only recently garnered experimental attention. Gradient-mediated patterning often occurs during division, growth and motility. Because gradients cannot form instantaneously, their establishment, interpretation, and decay must occur concomitant with these processes. Remarkably, in the midst of these cellular dynamics, patterning is essentially invariant from one embryo to another. Understanding this reproducibility of embryonic patterning seems impossible without knowing the biophysical parameters that give rise to gradient dynamics, as well as the degree to which the final developmental pattern reflects precision and reproducibility in the gradients themselves. Biophysical parameters of production, movement, and degradation of extracellular factors dictate the spatial and temporal range over which they can function. Such parameters can be measured in living tissue using fluorescently tagged proteins, e.g., molecular lifetime measured with reversibly photoactivated fusions, and effective diffusion determined over large timescales (on the order of minutes) by conventional fluorescence recovery after photobleaching. For low concentrations of quickly diffusing proteins, fluorescence correlation spectroscopy (FCS) offers an exciting alternate approach. FCS has been successfully applied to study rapid diffusion of FGF8 in zebrafish embryos and to examine receptor-ligand colocalization. Remarkably, such analyses yield estimates of in vivo ligand-receptor affinity (Ries et al., 2009Ries J. Yu S.R. Burkhardt M. Brand M. Schwille P. Nat. Methods. 2009; 6: 643-645Crossref PubMed Scopus (104) Google Scholar), which would be difficult or perhaps impossible to obtain from any living, intact context other than a developing embryo. Similar studies could be performed to capture the behavior of other extracellular signaling molecules, and any morphogen gradient will be better analyzed if its reproducibility is assessed by careful comparison of many samples using such technologies. Only by these approaches can we hope to understand whether and how an extracellular signal elicits precise responses in the face of dynamic cellular activities. Further, to determine how embryos achieve reproducibility, the dynamics of extracellular signaling molecules must be compared to the dynamics of the resulting cellular responses. Subsequent to extracellular signaling, patterning requires the correct intracellular transmission of extracellular cues. Advances in fluorescent imaging now provide unprecedented opportunities for visualizing intracellular signal transduction from the plasma membrane to the nucleus. Generally, these approaches utilize genetically encoded fluorescent reporters, usually Förster resonance energy transfer (FRET)-based, whose fluorescence spectra and/or emission intensities are altered upon signaling. Multiple reporters now exist to monitor the spatiotemporal control of, for example, second messenger molecules, G protein coupled receptors, and Ras activity (reviewed in Balla, 2009Balla T. Trends Cell Biol. 2009; 19: 575-586Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Excitingly, intracellular gradients of Aurora kinase and Ran GTPase have been visualized and demonstrated to regulate mitotic events and nuclear translocation activity (reviewed in Kholodenko et al., 2010Kholodenko B.N. Hancock J.F. Kolch W. Nat. Rev. Mol. Cell Biol. 2010; 11: 414-426Crossref PubMed Scopus (436) Google Scholar). Moreover, FRET sensors could be used to detect gradients in kinase activity of signal tranducers like protein kinase A or MAP kinase that play central roles in patterning. In principle, the activity of nearly any kinase can be measured by constructing a FRET sensor containing the peptide targeted for phosphorylation by the kinase of interest (Ni et al., 2006Ni Q. Titov D.V. Zhang J. Methods. 2006; 40: 279-286Crossref PubMed Scopus (62) Google Scholar). In addition to gradients of enzymatic activity, protein localization is also generally altered upon signaling (e.g., relocalization between nucleus and cytoplasm, or to the plasma membrane), and the dynamics of fluorescent protein fusions in living cells are highly amenable to quantitative analysis. Moreover, gradients of such intracellular activities may provide cells with directional knowledge for vectored motility, as well as for the establishment and maintenance of planar cell polarity. Therefore, the same FRET sensors may provide tools to follow the establishment and maintenance of molecular asymmetries that control spatially distinct behaviors. By documenting the dynamics of intracellular signal transduction in living embryos, it will also be possible to determine how cells tune their responses to the magnitude of the external signals they receive. Only by these methods can we determine the mechanisms that faithfully propagate externally supplied patterning information or that serve to filter noise, thereby ensuring that accurate positional information reaches the nucleus to control subsequent cellular dynamics. Upon receiving patterning information, the resulting program of gene expression in a cell must be appropriate to the magnitude and combination of signals received. Recent advances in cell imaging should allow us to determine how extracellular information is interpreted at the level of gene activity. Quantitative measurements of transcriptional output on a cell-by-cell basis are performed widely in single cell organisms and in mammalian cell culture (Darzacq et al., 2009Darzacq X. Yao J. Larson D.R. Causse S.Z. Bosanac L. de Turris V. Ruda V.M. Lionnet T. Zenklusen D. Guglielmi B. et al.Annu. Rev. Biophys. 2009; 38: 173-196Crossref PubMed Scopus (97) Google Scholar), and observations of mRNA dynamics can be combined with simultaneous measurements of transcription factor concentration to derive input-output relationships (Garcia et al., 2010Garcia H.G. Sanchez A. Kuhlman T. Kondev J. Phillips R. Trends Cell Biol. 2010; 20: 723-733Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Ideally, any study of cell fate specification should adapt similar methods to quantitatively describe gene expression as a function of transcription factor input concentrations. Developing embryos offer unique advantages to understanding gene expression modulation, namely the occurrence of multiple, overlapping combinations of input signals at physiologically meaningful magnitudes. The use of quantitative methods will allow a deep exploration of the promoter-enhancer architecture(s) necessary to confer transcriptional responses appropriate in time, space, and intensity. These approaches can be complemented by biochemical measurements of enhancer affinities and promoter occupancy. By relating transcription factor biophysical characteristics, such as DNA binding and polymerase recruitment, to transcriptional output observed in vivo, it should become possible to answer long-standing questions regarding the means by which graded, dynamic, and combinatorial inputs precisely and robustly regulate gene expression programs in time and space. In addition to gene expression, differential cell shape and motility also constitute cellular outputs to patterning cues. The previous decade has shown that the behaviors of large fields of cells can be characterized using quantitative image analysis. Yet, despite recent progress documenting the emergence of tissue-level behavior from the activities of individual cells, major questions remain unanswered regarding, for example, the mechanistic links between patterning and cell shape, as well as how molecular activity results in specific cellular behaviors. An essential first step in analyzing any morphogenetic process is to identify the cells actively engaged in force generation and the cytoskeletal elements and molecular motors within those cells responsible for that activity. One key challenge in the subsequent analysis is to connect those biochemical and biophysical properties to the generation of tissue-level behavior. Meeting this challenge will necessarily involve a multilevel approach, from measuring in vitro activities of purified factors to fluorescence microscopic observations of molecular dynamics on subminute (or even shorter) timescales to collecting morphometric data of entire developing organisms on the scale of hours. Although their adaptation to intact embryos presents substantial hurdles, biophysical analysis tools such as atomic force cantilevers and optical tweezers can potentially provide measurements of the magnitude of forces generated within cells and exerted on the extracellular environment. With this collection of approaches, studies of morphogenesis should aim to unify observations on a variety of time and length scales to thus understand how temporally and spatially restricted molecular activities give rise to the marvelous patterns of morphogenesis observed in diverse organisms. Our understanding of fundamental biological processes derives in large part from combining observation and manipulation of the underlying molecules. Recently emerged analytic tools now extend both our observation and manipulation capabilities. First, super-resolution microscopy methods afford unprecedented access to intracellular organization at a spatial resolution of only a few tens of nanometers, allowing visualization of subcellular-scale structures as never before (Toomre and Bewersdorf, 2010Toomre D. Bewersdorf J. Annu. Rev. Cell Dev. Biol. 2010; 26: 285-314Crossref PubMed Scopus (267) Google Scholar). In particular, structured illumination and stimulated emission-depletion can achieve super-resolution at substantial tissue depth, and so these approaches may be most amenable for use in embryonic contexts. Multicolor colocalization should be employed to explore the composition of multicomponent protein complexes in developing embryos, especially in cases where protein-protein interactions are difficult to analyze biochemically. Second, genetically encoded force reporters enable the direct observation of forces generated within living cells. In one innovative approach, a stretchable, elastic FRET reporter was fused to the head and tail domains of vinculin, a protein that binds to integrins and to actin filaments (Grashoff et al., 2010Grashoff C. Hoffman B.D. Brenner M.D. Zhou R. Parsons M. Yang M.T. McLean M.A. Sligar S.G. Chen C.S. Ha T. Schwartz M.A. Nature. 2010; 466: 263-266Crossref PubMed Scopus (920) Google Scholar). Cells lacking tension showed high FRET efficiency, whereas stretching of the molecule due to intracellular tension decreased FRET. Intracellular force measurements have the potential to bring long-sought answers to many fascinating but otherwise intractable questions regarding force generation and propagation in developing tissues. As such, many such reporters should be constructed and tested in embryonic contexts. Finally, optogenetics provides a startlingly powerful approach for manipulating molecular and cellular function at any desired temporal or spatial coordinate using light-sensitive factors derived from bacteria or plants. For example, when neurons are engineered to express a light-responsive membrane channel, they can be reversibly activated (or inactivated) upon exposure to blue light (Hegemann and Möglich, 2011Hegemann P. Möglich A. Nat. Methods. 2011; 8: 39-42Crossref PubMed Scopus (80) Google Scholar). The approach successfully modulates electrical activity in the cells of adult C. elegans (reviewed in Brown and Schafer, 2011Brown A.E. Schafer W.R. Nat. Methods. 2011; 8: 129-130Crossref PubMed Scopus (3) Google Scholar) and zebrafish embryos (Arrenberg et al., 2010Arrenberg A.B. Stainier D.Y. Baier H. Huisken J. Science. 2010; 330: 971-974Crossref PubMed Scopus (313) Google Scholar). Importantly, the method is not confined to studies of neuronal activity. Photoactivation has been used to control Cre activity in cultured cells and transcription in yeast (reviewed in Toettcher et al., 2011Toettcher J.E. Voigt C.A. Weiner O.D. Lim W.A. Nat. Methods. 2011; 8: 35-38Crossref PubMed Scopus (170) Google Scholar). Most excitingly, a photoactivatable Rac redirects the migration of neutrophils in zebrafish embryos (Yoo et al., 2010Yoo S.K. Deng Q. Cavnar P.J. Wu Y.I. Hahn K.M. Huttenlocher A. Dev. Cell. 2010; 18: 226-236Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) and of border cells in the Drosophila ovary (Wang et al., 2010Wang X. He L. Wu Y.I. Hahn K.M. Montell D.J. Nat. Cell Biol. 2010; 12: 591-597Crossref PubMed Scopus (229) Google Scholar), effectively demonstrating the power of optogenetic approach in vivo. In principle, this approach can be used to modulate nearly any factor of interest. The effects are reversible and confined spatially by restricted illumination. It may soon be possible to define the precise spatial and temporal domains over which a given factor is required to elicit a cellular response. The new experimental possibilities will likely require the reformulation of both our experimental approaches and our understanding of the molecular activities implicated in patterning and morphogenesis. In summary, the previous decade has witnessed rapid and significant progress in the application of biophysical and quantitative methods to the analysis of fundamental problems in cell and developmental biology. In our opinion, the diverse approaches we have discussed offer the promise of defining embryonic patterning as the result of coordinated biochemical activities. The continued integration of multiscale analyses of molecules, cells, and tissues is certain to open new frontiers in the understanding of the basic cell biologic processes underlying embryogenesis." @default.
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• W2026671350 title "Shifting Patterns: Merging Molecules, Morphogens, Motility, and Methodology" @default.
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