The transformation of the model organism: a ... - Semantic Scholar

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The transformation of the model organism: a decade of developmental genetics Kathryn V. Anderson1 & Philip W. Ingham2

© 2003 Nature Publishing Group http://www.nature.com/naturegenetics

doi:10.1038/ng1105 The past decade has seen the development of powerful techniques to dissect the molecular processes that regulate development. New tools have been used to reveal the basis of cell polarity, morphogen gradients and regulation of signaling in developing animals. Cell biology and developmental biology have become closely intertwined, and many genes that had been thought of as regulators of general cell biological (housekeeping) functions have been shown to act as specific developmental regulators. Vertebrate developmental genetics is now flourishing, with forward and reverse genetics in both zebrafish and the mouse providing new dimensions to our understanding of development.

A revolution took place in the field of developmental biology between 1982 and 1992. The convergence of classical and molecular genetics, largely in Drosophila, transformed the mystery of development into a biological phenomenon that could be described at a satisfying molecular level. By 1992, the outlines of processes as complex as axis specification, segmentation and neurogenesis were known in Drosophila. Molecules that act as localized cytoplasmic determinants had been discovered. Hox, zinc-finger and bHLH (basic helix-loop-helix) family transcription factors had been identified as regulators of key cell fate decisions. The concepts that extracellular ligands could act as morphogens or as switches were established. Components of several signaling pathways had been identified and primary functions of these pathways defined. Most importantly, there were key examples, like the Hox clusters, that suggested that these molecules could have conserved functions in evolution. In 1994, Lewis Wolpert1 summarized the improved status of developmental biology: “In general…it can be argued that the principles of development are understood, although many crucial details at the molecular level are missing. For example, there is not a single case in all of vertebrate development where an intercellular signal has been unequivocally identified… We also still do not know how signals are propagated and whether there are…graded distributions of diffusible morphogens. The downstream targets of the Hox genes remain elusive… We remain largely ignorant of timing mechanisms and how size is regulated…” What have we learned in the intervening years? One surprise that has emerged is that a rather small number of signaling pathways control developmental decisions, and there is extraordinary diversity in the responses of cells to those signaling pathways. Other genuine insights have come from the twists of the logical circuitry of those signaling pathways. As Wolpert foresaw, the development of powerful new techniques has revealed many

details about the molecular processes that underpin development, at a level that could not have been anticipated a decade ago. We now have complete genome sequences, colored proteins that we can observe in vivo and the ability to express any gene at any time or place in whole organisms. Many of the findings of the past decade, both the new logical insights and the details of particular developmental processes, have surprising importance for human health. Who would have guessed ten years ago that we would learn about the molecular mechanisms of aging, dementia and immune responses by studying embryonic development in flies and worms? Perhaps the greatest advance in the past ten years has been the growth of molecular understanding in the development of vertebrate embryos. Powerful new genetic approaches in both zebrafish and the mouse have not only identified intercellular signals, but also elucidated how those signals act in many different contexts. Here we provide an overview of progress in the last decade, highlighting examples of fundamental insights derived from worm and fly genetics and progress in vertebrate developmental genetics.

A decade of development Visualizing cell and tissue polarity Wolpert recognized that many developmental asymmetries are derived from cellular asymmetry. Much was learned about cell polarity in the ensuing decade, stimulated by high-resolution imaging afforded by confocal microscopy and by the ability to visualize proteins in living cells and embryos, made possible through the discovery and development of green fluorescent protein (GFP)2. The early development of Caenorhabditis elegans is characterized by a series of asymmetrical divisions that generate the six founder cells of the embryo. These divisions depend critically upon the activity of the par genes3, the protein products of which localize to the cortex of the embryo, PAR1 and 2 at its posterior

1Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. 2Centre for Developmental Genetics, Department of Biomedical Science, University of Sheffield, Sheffield, UK. Correspondence should be addressed to K.V.A. (e-mail: [email protected]).

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Fig. 1 Cell and tissue polarity revealed by GFP fusions. a, Time-lapse imaging of PIE-1:GFP distribution during the first cell cycle of a living C. elegans embryo4, showing the progressive segregation of PIE-1 from an initial uniform distribution in the cytoplasm of the one-cell embryo to the nucleus of the posterior blastomere, the precursor of the germ line. The left-hand series shows the embryos in Nomarski optics, and the right-hand panel shows GFP expression in the same embryos. b, Frizzled:GFP in wild-type wing cells73. Frizzled:GFP accumulates in a point on the distal side of each cell. Green represents Frizzled:GFP; red represents the actin cytoskeleton. c, Localization of Frizzled:GFP depends on the product of the gene inturned. In the inturned mutant, the position of Frizzled is variable.

end and PAR3 and 6, along with atypical protein kinase C (aPKC), at its anterior end. One effect of localized PAR activity is to displace the first cleavage spindle posteriorly, resulting in cleavage of the one-cell embryo into two daughters of unequal size. At the same time, the PIE-1 protein—a CCCH finger protein essential for development of the germ line—is preferentially distributed to the posterior daughter, from which the germ line is derived. To understand the basis of this asymmetrical distribution, Reese et al.4 generated embryos expressing a functional PIE1:GFP fusion protein and used time-lapse confocal microscopy to follow its distribution during the early cleavage stages (Fig. 1a). These studies revealed that PIE-1 is initially distributed uniformly throughout the cytoplasm of the embryo, but becomes enriched at the posterior end and depleted at the anterior end before cell division in response to PAR1 activity. They also identified regions of the protein necessary and sufficient for this pattern of accumulation, opening the way to determining the precise molecular mechanism by which PIE-1 is regulated by PAR1 serine/threonine kinase. In Drosophila, neuroblasts divide unequally, regenerating themselves and giving rise to neuronal precursors known as ganglion mother cells (GMCs). This asymmetrical division is associated with the asymmetrical segregation of two protein complexes necessary 286

for GMC fate—the Miranda complex, which includes the homeodomain protein Prospero, and the Numb complex, consisting of Numb itself, an inhibitor of Notch activity, and the adaptor protein, Partner of Numb (Pon), which is required for the subcellular localization of Numb. As in the early worm embryo, PAR3 and PAR6 are crucial in the asymmetrical divisions of the neuroblasts5. Both proteins, along with aPKC, localize, in this case not to one side of the cell but to its apical surface where they recruit two further proteins, Insc and Pins, not found in C. elegans. All five proteins are required for the localization of the Miranda and Numb complexes to the basal cortex of the cell. Insights into how the Numb complex is localized have come from in vivo imaging of a Pon:GFP fusion protein. Using time-lapse confocal microscopy analysis, Lu et al.6 found that Pon is initially recruited from the cytosol to the cortex and then moves around the cortex, eventually becoming restricted to the basal side. The functions of PAR3, PAR6 and PKC3 seem to have been highly conserved in evolution. In mammals, their orthologs ASIP, mPAR6 and aPKC form a complex that localizes to tight junctions in epithelial cells and is implicated in their formation and the specification of apical basal polarity7. An independent system of cell polarity depends on activity of the Frizzled (Fz) protein rather than on PAR proteins. Frizzled was originally identified through its effects on the polarity of ectodermal cells; for instance, in the Drosophila wing, Fz activity is required for the correct polarity of the hairs secreted by each cell of the wing blade. GFP fusions and confocal microscopy8,9 have revealed that the distribution of Fz is itself polarized in each cell, as are the Dishevelled and Flamingo proteins, which act downstream of Fz in the so-called planar cell polarity (PCP) pathway (Fig. 1b,c). Misexpressing morphogens After a decade focused on the embryogenesis of Drosophila, the 1990s saw a renaissance of the imaginal disc as a model system for the analysis of growth and pattern. Crucial to these studies has been the application of high-resolution imaging techniques to visualize the distribution of proteins across these epithelial sheets, in particular the secreted proteins encoded by the genes hedgehog (hh), decapentaplegic (dpp) and wingless (wg). In parallel, breakthroughs in manipulating gene expression in imaginal discs that used regulatory genes from yeast made it possible to test the functional significance of these protein distributions. The ‘flip-out’ technique developed by Basler and Struhl10 takes advantage of the demonstration by Golic and Lindquist11 that the yeast Flip recombinase can catalyze site-specific recombination in Drosophila carrying the appropriate FLP recognition target (FRT) sites. This ingenious methodology allows a gene of interest to be activated in a clone of cells in an otherwise wild-type animal, while simultaneously marking the clone with a gratuitous visible marker, such as the yellow gene that controls cuticle pigmentation (Fig. 2a). The second technique, developed by Brand and Perrimon12, is based upon the demonstration by Fischer et al.13 that the yeast transcription factor GAL4, although normally silent in Drosophila, can direct transcription from promoters containing the GAL4 binding sites known as upstream activating sequence (UAS). Brand and Perrimon used the enhancer trapping technique14 to generate a large library of lines in which expression of GAL4 is driven in specific temporal and spatial patterns by endogenous enhancers. Crossing such ‘driver’ lines to others carrying genes of interest cloned downstream of the UAS generates animals in which a given gene is misexpressed in a defined place and time during development. Using these new genetic tools, several labs have defined the roles of the three key signaling proteins, Hh, Dpp and Wg, in nature genetics supplement • volume 33 • march 2003

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Fig. 2 Stable activation of gene expression using the flp-out technique. a, The yeast flp recombinase mediates site-specific recombination between cis-acting FRT sites (arrowheads). In the flp-out cassette, two FRT sites flank the coding region of a marker gene, such as yellow (green line), and a transcriptional terminator signal. This is placed downstream of a constitutive promoter and upstream of the gene of interest. In the absence of recombination, expression of the marker gene is driven by the constitutive promoter, and the gene of interest remains silent. After FLP-mediated recombination, the marker gene and associated transcription termination signal are removed from the cassette, placing the gene of interest under control of the promoter. b, Example of a clone of Hh-expressing cells (green) induced by the flp-out technique. Hh activity emanating from this clone induces expression of the Hh target genes ptc (blue) and dpp (red) in cells at different distances from the boundary of the clone.

organizing fields of imaginal disc cells into specific patterns15–18. In each case, these experiments demonstrated that the signaling proteins act in a concentration-dependent manner to specify the identity of individual cells (Fig. 2b). Moreover, tethering the proteins to the surface of expressing cells demonstrated that each protein acts directly and at a distance to effect the specification of cell fate, a fundamental property of a classic morphogen. Although the mechanisms that generate morphogen gradients are still topics of passionate debate, the data derived from these experiments and from the spatial distributions of Hh, Dpp and Wg proteins have been detailed enough to derive mathematical models of how stable morphogen gradients might form in the wing disc19.

New regulators through clonal and modifier screens The reemergence of the Drosophila imaginal disc as an experimental paradigm has been driven also by the development of an efficient method of inducing mitotic recombination that circumvents the limitations of X-ray–mediated somatic crossing-over. Xu and Rubin20 introduced FRT sites close to the centromeres of each of the major chromosome arms, allowing the induction of clones simply by producing short pulses of flippase activity expressed under the control of the inducible heat-shock promoter (Fig. 3a). Being able to generate clones of cells homozygous with respect to whole chromosome arms at high frequency opened the way to screening for mutations in mosaic animals, a highly efficient way of identifying genes required throughout development.

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Fig. 3 Induction of labeled clones of mutant cells using FRT-mediated somatic recombination. a, The circle on the left represents a cell heterozygous with respect to a mutation (m), and a transgene that drives constitutive expression of a gratuitous cell marker gene, such as GFP (green triangle), at the four-strand stage of the cell cycle. Induction of FLP activity at this stage promotes recombination between the centromere-proximal FRT sites. Segregation of the recombinant chromosomes at mitosis generates two daughter cells, one of which is homozygous for the mutation and lacks the marker transgene, and one which is homozygous wild-type and carries the transgene. The resulting homozygous mutant clone can thus be identified on the basis of its lack of marker gene expression. b, Examples of clones homozygous with respect to a mutation in pka and lacking expression of the marker gene that is expressed in all other cells (left panel). Note that the wild-type twin clones carry two copies of the marker and therefore express it at higher levels than the surrounding heterozygous cells. Cell-autonomous accumulation of the Ci transcription factor, resulting from inactivation of PKA74 (right panel). Figure courtesy of the Company of Biologists.


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review Notable examples of genes discovered in clonal screens include several that encode proteins involved in regulating Hh signaling. Two of these, protein kinase A and Slimb, have been implicated in the proteolytic processing of the transcription factor Cubitus interruptus (Ci), a process that transforms the full-length activating form of this transcription factor into a transcriptional repressor21,22. Clones of cells mutant with respect to either gene accumulate full-length Ci protein (Fig. 3b), which activates Hh target genes inappropriately, causing patterning abnormalities in the imaginal discs. A different approach to identifying components of signal transduction pathways is to screen for enhancer and suppressor mutations. This strategy was applied exhaustively to the analysis of receptor tyrosine kinase signaling in Drosophila, taking advantage of the nonessential nature of the fly eye. The gene sevenless encodes a receptor tyrosine kinase required for specification of one of the eight photoreceptor cells in each of the 1,000 ommatidia that make up the compound eye. By screening for modifiers of the sevenless phenotype, Rubin, Simon and colleagues identified several genes encoding components of the RTK-Ras-RafMAP kinase pathway23,24. The success of this approach has inspired many other successful genetic modifier studies. Negative feedback loops A seemingly universal rule emerged with the discovery of more of the components of intercellular signaling pathways: signaling pathways activate negative feedback loops that limit the extent or spread of the response to signals25,26. Each of the major developmental signals activates transcription of at least one negative regulator of its pathway (Fig. 4). This rule is so robust that Gerlitz and Basler27 were able to use gene expression patterns to identify new negative feedback regulators of the Wingless signaling pathway. Some signals, like Drosophila Wingless, have more than one negative feedback regulator. Naked and Wingful are both transcriptional targets of Wg signaling whose feedback inhibits two distinct steps in the Wg pathway. Naked acts intracellularly, binding to Dishevelled and blocking the accumulation of Armadillo (β-catenin) in response to Wg, whereas Wingful is secreted and acts nonautonomously. These two types of inhibitors apparently constrain signaling in two different dimensions: time and space. From signaling pathways to cell biology As a result of what has been learned from modifier and clonal screens, a new view is emerging about the nature of the components that regulate intercellular communication during development. In the first wave of discovery of developmental regulatory genes, it seemed that all the regulators were either transcription factors or ligands and receptors. This engendered a view of communication between cells as a simple process that involves the production of a ligand by one cell, binding of the ligand to the

Fig. 4 Negative feedback loops restrict the spatial and temporal range of signaling molecules. Each of the major signaling pathways activates the transcription of one or more genes that encode negative regulators of the signaling pathway. If the negative regulator inhibits signal transduction within the cell, it is likely to shut down signaling with time. If the negative regulator acts outside the cell, it can limit the spatial spreading of the signal.

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receptor on another cell, and activation of a transcription factor in response to ligand binding. Although this early picture provided a valuable framework, as new genetic screens identified more of the components required it became clear that developmental decision-making depends upon an exceptional diversity of protein functions. Many of the newly identified molecules control the cell biological environment in which cells communicate. Dedicated proteins control the secretion of specific ligands, while extracellular components promote and constrain their diffusion. Protein modification and protein turnover are also key steps in the production and response to signals. Cytoplasmic responses to signals depend on protein machines that are localized in specific compartments in the cytoplasm or nucleus. Some signaling processes bypass the nucleus and act directly on the cytoskeleton and extracellular matrix. In many cases, mutations in these ‘cell biological’ factors produce remarkably specific mutant phenotypes, overthrowing the traditional concept of ‘housekeeping’ genes. Indeed, given the reiterative use of a relatively small number of signaling molecules (Wnt, Bmp, Hh), much of the specificity in response to signals comes from these cell biological modifiers. Dozens of proteases have been identified that have specific roles in activation or degradation of signaling components. Processing proteases can activate signaling components outside the cell (such as in the specification of the Drosophila dorsal–ventral axis), inside the cell (as in Hedgehog signaling) or in the plane of the membrane (such as Notch)28. Ubiquitin-mediated protein degradation is an integral step in the Notch29, TGFβ30, Wnt and Toll signaling pathways31. Degradation also can be modulated in response to signals. For example, lysosomal degradation of endocytosed Wg protein is regulated by epidermal growth factor receptor (EGFR) activity, and that determines the spatial distribution of active Wg32. Dedicated proteins have been found to regulate ligand secretion. Secretion of the Drosophila TGFα protein Spitz requires the sequential activity of two proteins. Star is present throughout the secretory pathway and is necessary to export Spitz from the endoplasmic reticulum to the Golgi apparatus33. Rhomboid-1, a seven-span transmembrane protein with serine protease activity, is localized in the Golgi, where it promotes the intramembrane cleavage of Spitz. The Drosophila Windbeutel protein is required for the Pipe protein to move from the endoplasmic reticulum to the Golgi, where it can be appropriately modified for activity34. Pipe is a heparan sulfotransferase that modifies an unknown glycosaminoglycan required to activate proteolytic processing of the ligand Spätzle35. The putative acyltransferase encoded by the gene porcupine controls Wg secretion, apparently by facilitating its glycosylation36. Secretion of Hedgehog depends upon the activity of Dispatched, a multipass transmembrane protein of the Patched/CHE14/NDR family37.

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Fig. 5 Covalent modification is one mechanism used to modulate the activity of proteins required for intercellular signaling. The Notch receptor is covalently modified by the action of by at least eight different genes; some modifications promote its activity, and others restrict its activity. Some modifications occur in all cell types, and some are cell type–specific.

The activity of receptors is also controlled by their cellular environment. Presentation of the Slit receptor Robo on the surface of axonal growth cones depends upon the activity of Commisureless (Com), a newly discovered transmembrane protein that acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway38. The Notch pathway takes the use of covalent modification to the extreme39 (Fig. 5). Fringe regulates O-fucosylation of the Notch extracellular domain, which controls which ligand can activate Notch in inductive interactions40. Two different proteases proteolytically cleave Notch: Kuzbanian is required to allow ligand activation, and ligand binding leads to proteolytic cleavage of Notch within the plane of the membrane by Presenillin, which generates the active Notch fragment that moves to the nucleus to activate transcription. The cytoplasmic domain of Notch can be ubiquitinated at different sites, regulated by different E3 proteins: Neuralized, Suppressor of deltex, Sel-10 and Itchy. Each of these modifications has the potential to be specific to particular aspects of Notch function or particular cell types. Once a receptor is activated, intracellular trafficking of the ligand–receptor complex and downstream signaling components is vital. Ubiquitination of the EGF receptor is required for endocytosis of the ligand–receptor complex, which in turn modulates signaling by that complex. Rab23 and the kinesin-like molecule encoded by costa act as specific negative regulators of Hedgehog signaling41,42. Despite the links that have been made between development and cell biology, a number of common processes intimately involved with both cell and developmental biology are not understood. For example, morphogenesis, or the processes that control how cells and tissues change shape, remains largely enigmatic. Visualization of cells and molecules in wild-type and mutant animals will be important tools in dissecting morphogenesis, but much work remains ahead. From worms and flies to vertebrates: triumphs of reverse genetics Perhaps the biggest change in the character of developmental genetics is that vertebrate embryos are now legitimate targets for in-depth genetic analysis. Ten years ago, the basic techniques for disrupting genes in the chromosome by homologous recombination in embryonic stem (ES) cells had been developed, but had been used only to disrupt a handful of genes important in development (Wnt1, two Hox genes, and En2). Now, approximately 3,000 mouse genes (roughly 10% of the genes in the genome) have been disrupted using ES cell technology. Moreover, refinements of ES cell technology have led to conditional alleles, tissuespecific knockouts and alleles that knock in specific point mutations, which have made it possible to dissect the functions of genes used repeatedly in different developmental contexts. A particularly sophisticated example of ES cell manipulations was developed by Duboule and colleagues43 and used in the nature genetics supplement • volume 33 • march 2003

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analysis of transcriptional regulation of the Hox gene clusters. By driving expression of the yeast Cre recombinase under the control of a synaptonemal complex gene promoter, unequal crossing-over between homologous chromosomes can be induced, generating deletions and duplications of specific loci. Using this approach, Knita et al.43 described the different phenotypic effects of a conventional targeted mutation of a gene and its physical deletion, a disparity that highlights the importance of position for correct transcriptional regulation. These analyses revealed how a distantly located ‘digit’ enhancer acts over a long distance to regulate the transcription of genes at the 5′ end of the HoxD cluster, giving new insights into the mechanistic basis of digit formation in the vertebrate limb. Much of the speed of our progress in understanding vertebrate development has come from the conservation of the small set of signaling pathways defined in flies and worms and the ability to target the inactivation of these genes in the mouse using ES cell technology. Targeted mutations have provided a framework for examining the series of tissue interactions that control mammalian embryogenesis (and vertebrate embryogenesis as a whole). Analysis of Shh signaling, for instance, has provided one of the best examples so far of a putative morphogen activity in a vertebrate embryo, whereas analysis of mutations in the Wnt and Notch signaling pathways have given insights into processes as diverse as development of hair follicles and T lymphocytes44,45. Crucial to the analysis of the functions of these genes have been the advances made in analyzing the mouse embryo. A simple technical innovation, the application of whole-mount in situ hybridization46 helped show that the mouse body plan is similar to that of other vertebrate embryos (Fig. 6). At the same time, experimental manipulation of the mouse embryo led to the identification of important organizing centers that control the elaboration of the body plan. In a landmark 1994 experiment, Rosa Beddington transplanted the node from one mouse embryo to another and showed that, as with Hensen’s node in the chick or Spemann’s organizer in the frog, the node was sufficient to induce an ectopic body axis47. These experiments also revealed something surprising: although the trunk was duplicated, the head was not. This led to the conclusion that something besides the node organized the head of the mouse embryo. Beddington followed up with experiments that defined a group of cells that 289


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help organize the head in the anterior visceral endoderm (AVE). These extraembryonic cells are important in establishing the anterior–posterior axis of the mouse embryo48. The identification and functional analysis of genes specifically expressed in the AVE showed that the mouse embryo could lead the way in some aspects of vertebrate embryology. Efficient and rapid approaches to inactivating genes of known sequence have been developed and successfully applied in other vertebrate systems, notably the zebrafish. Antisense morpholino–modified oligonucleotides designed to block translation or splicing of specific mRNAs can be used to knock down the function of genes identified on the basis of their sequence49. Injection of such oligonucleotides into newly fertilized eggs has highly specific effects on a wide variety of genes. This opens the way to the rapid functional analysis of newly identified genes while simplifying the generation of double- or even triple‘mutant’ embryos. RNA interference (RNAi) approaches also show some promise for providing similar rapid gene inactivation in the postimplantation mouse embryo50,51. Forward with genetics: from flies to fish... and mice Notwithstanding the power of reverse genetics, the case for carrying out forward genetic analysis of vertebrate embryogenesis has become increasingly appealing after the success of the Drosophila and C. elegans screens. The zebrafish—with its rapid development, easily accessible and transparent embryos and prodigious fecundity—has found favor with many investigators as a kind of vertebrate Drosophila. At the beginning of the 1990s, the number of zebrafish mutations affecting embryonic development could be counted on the fingers of one hand—yet it now exceeds 8,000 and is continuing to rise. As was the case with Drosophila a decade earlier, the principal driver of this explosion of experimental material has been systematic mutant screens

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Fig. 6 Whole-mount in situ hybridization makes it easier to visualize the anatomy of the mouse embryo. a, Brachyury (T) RNA is enriched in the tail bud and notochord of the 10.5day embryo75 Figure courtesy of the Company of Biologists. b,c, Left–right asymmetry is detectable in the 8.2day mouse embryo, as visualized by the expression pattern of Nodal (b) and Lefty1 (c). The gene Nodal is expressed in the node and the left lateral plate mesoderm; Lefty1 is expressed in the midline and the left lateral plate mesoderm76.

carried out by Nüsslien-Volhard and colleagues in Tübingen, Germany and mirrored by Driever, Fishman and colleagues in Boston, Massachusetts, USA. A central rationale for studying the zebrafish was to apply the same type of analytical approach used so successfully in invertebrate models to the analysis of processes and structures that characterize the developing vertebrate embryo. In this respect, the screens have been highly successful, as evidenced by the isolation of mutants with disruptions in everything from gastrulation or somitogenesis to neural crest migration or blood cell differentiation (Fig. 7). In some cases, molecular analysis of these mutants—such as those with disruptions in dorso-ventral patterning of the embryo—has confirmed the roles of specific signaling pathways worked out through different approaches in other organisms, especially Xenopus laevis. In other cases, analysis of zebrafish mutants has provided truly new insights. The one eyed pinhead mutant, for instance, revealed a role for the previously enigmatic Cripto protein in mediating receptor activation by Nodal, a member of the TGFβ superfamily of secreted proteins52. The development of the heart has attracted particular attention in the zebrafish, owing to the ability of the embryo to survive for several days simply on diffused oxygen. This means that mutants lacking a functional heart do not have complex pleiotropic phenotypes, greatly facilitating their identification. Many such mutants have been isolated, and the molecular characterization of some of these has already provided important new insights into the development of this vital organ. The miles apart mutation, for instance, inactivates the sphingosine-1-phosphate receptor, revealing an unexpected role of lyposphigolipids in regulating migration of the cardiac progenitors53. The jekyll mutation, which inactivates a UDP glucose dehydrogenase required for glycoasminoglycan synthesis, has shed new light on the signaling processes controlling valve formation54. Molecular cloning is currently a rate-limiting factor in the analysis of genes identified in chemical mutant screens, but the application of single-nucleotide polymorphism (SNP) mapping, made possible through the genome sequencing project ongoing at the Sanger Institute, is set to accelerate the cloning of zebrafish genes defined by ENU-induced mutations. In the same way, completion of the mouse genome sequence has made positional cloning in this organism straightforward. As a result, chemical mutagenesis and forward genetic screens in the mouse are beginning to identify the functions of previously Fig. 7 Identification of mutations in zebrafish. a,b, Wild-type zebrafish larva four days after fertilization of the egg. Changes in morphology of the animal are easily detected by visual inspection, as exemplified by the larva homozygous for a null mutation in shh shown in b. Transcriptional control of genes can readily be visualized and analyzed in the zebrafish through generation of transgenic lines. c, An example showing 1-day embryos expressing GFP under the control of promoter elements from shh. Photographs courtesy of S. Roy and F. Muller.

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Fig. 8 Two strategies for forward genetics in the mouse identify new genes important in development. a–d, The gene Lrp6 was identified by a gene trap insertion mutation that causes a variety of defects, including loss of tissue at the mid/hindbrain boundary, similar to that seen in Wnt1 mutants77. a,c, Wild-type embryos (a, embryonic day (E)10.5; c, E14.5); b,d, mutant embryos. Molecular and phenotypic analysis showed that Lrp6 encodes a Wnt co-receptor. e,f, The open brain (opb) gene was identified in an ENU mutagenesis screen, based on defects in neural tube closure and eye differentation. e, Wild-type E10.5 embryo; f, opb E10.5 embryo (copyright (1998) National Acadamy of Sciences, U.S.A.)78. Molecular and phenotypic analysis showed that opb encodes Rab23, which acts as a negative regulator of Sonic hedgehog signaling42.

uncharacterized genes42,55–59. Elegantly designed gene trap experiments have provided another approach for forward genetics and have identified previously uncharacterized molecules based on mouse phenotypes60. More than 8,000 gene trap lines have been constructed61. Although many of these trapped ES cell lines have not yet been made into mice, they provide a valuable resource of mutations in genes of known sequence. Thus, forward genetics is now an important part of the repertoire of the mouse developmental geneticist (Fig. 8).

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What’s next? Connections to human health In ways that were not predictable ten years ago, model organisms have changed the study of human biology. A great number of developmental regulators discovered in Drosophila and C. elegans are important factors in human genetic disease, and with complete genome sequences available in many organisms, we have the dictionary to translate between organisms. Because of the evolutionary conservation of developmental regulators, we have learned about the molecular basis of a number of human birth defects. A particularly good example is human holoprosencephaly62, the absence of ventral parts of the brain and face. These structures are specified by Sonic hedgehog and Nodal (TGFβ family) signals, and mutations in a number of genes in these pathways have been associated with human holoprosencephaly. As understanding of the developmental events that control organ development becomes more comprehensive, it likely that the molecular bases of many birth defects will be clarified. It is now axiomatic that genes that regulate development also regulate the development of tumors, but that was by no means clear ten years ago. The first direct connection between developmental signaling pathways and cancer was made by a confluence of human genetics, molecular biology and Drosophila genetics. The adenomatous polyposis coli (APC) gene was identified by positional cloning of a heritable human predisposition to colon cancer. As APC was a new protein, clues to its function came from identification of interacting proteins, including β-catenin. At about the same time, β-catenin (known as Armadillo in Drosophila) was characterized as an essential component of the Drosophila Wnt pathway, suggesting that aberrant Wnt signaling might be involved in colon cancer. We now know that the Wnt signaling pathway promotes proliferation of stem cells in crypts in the colonic epithelium63, suggesting that tumors develop when there is inappropriate Wnt signaling in this cell type. A second example of the role of developmental regulators in cancer cemented the case. Patched, the receptor in the Hedgehog signaling pathway, has the unusual property of keeping the signaling pathway off in the absence of ligand, so that Patched mutations cause inappropriate activation of Hedgehog target genes. When a human ortholog of Patched was identified, it was mapped near a human tumor suppressor, the Gorlin syndrome gene, and mutations in human PTCH (patched homolog) were identified in individuals with Gorlin syndrome64. Thus, inappropriate activity of the Hedgehog pathway promotes certain types of tumors. nature genetics supplement • volume 33 • march 2003

Developmental genetic studies have also shed light on many aspects of human biology that seem to be far removed from development. For example, Toll, the transmembrane protein discovered and characterized because of its essential role in patterning the Drosophila embryo, is the founding member of the TLR family, crucial receptors in innate immune responses in both flies and mammals65. Notch is proteolytically processed by an enzyme that is important in Alzheimer disease66. Studies of the insulin signaling pathway in development led to studies of that pathway in aging (see ref. 67). These connections to human health are likely to become more and more central to developmental biology. Developmental regulators are reused repeatedly, not only during development but also in the replacement of tissues in the adult and in response to injury and infection. As a result, the fields of developmental biology and pathology now overlap significantly, and the two fields are likely to become tightly intertwined in the future. As we understand how developmental regulators are used in this particular developmental context, new targets for intervention in disease will be defined. Of course, all hopes for using stem cells to replace diseased cell types will rely on a deeper understanding of the basic developmental biology of stem cells. The death and transfiguration of the model organism The completed genome sequences in C. elegans, Drosophila, human, mouse, and, soon, zebrafish make it clear that we have much more to learn about the genes that control development. Even in Drosophila, functions have been assigned by mutations to only about 30% of the genes. The development of systematic, genome-wide RNAi, first in C. elegans68,69, will undoubtedly have a growing role in understanding gene function in all aspects of life, not just in developmental biology. Genome-wide expression analysis will also be important in identifying new functions of developmental genes70. The rigor and power of phenotype-based 291


review genetic analysis in flies and worms will continue to lead to new insights in basic, evolutionarily conserved processes. With the connections to human biology being made nearly as fast as a computer link, studies in worms and flies will be integrated into the analysis of most aspects of human biology, doing away with the old concept of ‘model organisms’ that are only theoretically relevant to human health and pathology. In the past decade, most developmental studies have focused on a few standard organisms that were selected for their odd lifestyles— particularly the ability to grow in the lab with a rapid generation time. As a result, it is likely that important biological processes have been overlooked that are better studied in other animals. Until recently, those interested in nonstandard organisms and in understanding the evolution of different developmental strategies were limited to describing patterns of gene expression. Now, with the application of RNAi71 and transformation techniques72 to more species, evo–devo (evolution and development) studies will be able move from description to mechanism, and models for new aspects of biology should bloom. Is it acceptable now to work on apparently arcane aspects of biology in model organisms without making direct connections to human health? It must be, because the best investigators recognize interesting questions that don’t fit into a predefined paradigm and follow the biology for its own sake. These curiosity-driven experiments are the ones that lead to truly surprising discoveries. We can expect that studies of seemingly exotic developmental events will continue to provide new perspectives on evolution and human biology. URL. Sanger Institute, http://www.ensembl.org/Danio_rerio/ 1. 2. 3. 4.

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