Milestones in Developmental Biology

Milestones in Developmental Studies (from Nature)

Milestone 1 (1924): Organizing principles – Barbara Marte Milestone 2 (1929): Taking a leaf from the book of cell fate – Natalie DeWitt

Milestone 3 (1937): Inhibit thy neighbour – Katrin Bussell Milestone 4 (1952): Symmetry breaking and computer simulation –Chris Surridge

Milestone 5 (1952): Turning back time – Nick Campbell Milestone 6 (1957): Out on a limb – Heather Wood

Milestone 7 (1963): Common sense – Jon Reynolds Milestone 8 (1969): Shaping destiny – Amanda Tromans

Milestone 9 (1971): Reinventing reproduction – Emma Green Milestone 10 (1977): Systems biology ahead of its time – Magdalena Skipper

Milestone 11 (1978): Order: it's in the genes – Tanita Casci Milestone 12 (1980s): Tools of the trade – Alison Schuldt

Milestone 13 (1980): How the fruit ﬂy gets its stripes – Rebecca Barr Milestone 14 (1981): A direct link – Rachel Smallridge

Milestone 15 (1986): 131 corpses and a Nobel prize – Marie-Thérèse Heemels Milestone 16 (1986): morphing heads – Sarah Greaves

Milestone 17 (1987): Two faces of the same coin – Sowmya Swaminathan Milestone 18 (1988): An unequal divide – Deepa Nath

Milestone 19 (1989): Chasing the elusive inducer – Annette Markus Milestone 20 (1990): The keys to sex – Myles Axton

Milestone 21 (1991): Sonic hedgehog, the morphogen – Jack Horne Milestone 22 (1995): Coordinating development – Louisa Flintoft

Milestone 23 (1995): A pathway to asymmetry – Kyle Vogan Milestone 24 (1997): Time for segmentation – Magdalena Skipper Milestone 1 (1924)

1 July 2004 | doi:10.1038/nrn1449 Organizing principles Barbara Marte, Senior Editor, Nature A central question in developmental biology is how form and pattern emerge from the simple beginnings of a fertilized egg. How and when do individual cells and tissues decide which developmental route to take? Are cell fates somehow predetermined or do cells and tissues interact with one another to orchestrate developmental processes? A groundbreaking (and technically demanding) experiment published in 1924 recognized one fundamental principle during development: embryonic induction. It provided the first unambiguous evidence that cell and tissue fate can be determined by signals received from other cells. This experiment, probably the best known in embryology, was carried out by Hilde Mangold, a Ph.D. student in the laboratory of Hans Spemann in Freiburg. The experiment was performed in newt embryos at the gastrulation stage, the period during which the three primary germ layers — ectoderm, mesoderm and endoderm — are © Images courtesy of established. It involved transplantation of a The International Journal structure on the dorsal side of the blastopore of Developmental Biology, UBC Press, Spain. embryo, called the dorsal lip, to the ventral side of another embryo. By grafting tissue between differently pigmented Triton species, the fates of the graft and host tissues could be distinguished. This graft, nowadays referred to as the Spemann organizer (also the Spemann–Mangold organizer) had two effects. It induced the formation of neural tissues from ectoderm that would have otherwise assumed an epidermal fate (neuralization), and it caused dorsalization of the ventral mesoderm, leading to the formation of somites. This resulted in the formation of a second embryonic axis, and therefore a twin embryo, at the graft site. Importantly, all of these structures were composed of both graft and host cells. This experiment therefore demonstrated the existence of an organizer that instructs both neuralization and dorsalization, and showed that cells can adopt their developmental fate according to their position when instructed by other cells.

The molecular nature of this signal remained In my opinion this is elusive until 65 years later (see also probably the single most Milestone 19), and it is still not completely influential research paper understood. In contrast to expectations, for to have been published in the field of developmental example, the signal released by the organizer biology. to cause dorsalization turned out not to be a direct inducer, but instead consisted of Ivor Mason antagonists, which block other inhibitors that are present throughout the mesoderm and that prevent the dorsalization of adjacent tissue. Hans Spemann was awarded the Nobel Prize in 1935 for his work on embryonic induction. Hilde Mangold tragically died in a house fire at the age of 26, even before the paper was published in 1924. Today, we know that their discovery of signals from one cell or tissue instructing the fate of others is a widely used principle in development.

REFERENCES ORIGINAL RESEARCH PAPER Spemann, H. & Mangold, H. Über induktion von Embryonalagen durch Implantation Artfremder Organisatoren. Roux' Arch. Entw. Mech. 100, 599–638 (1924)

FURTHER READING Sander, K. & Faessler, P. E. Introducing the Spemann–Mangold organizer: experiments and insights that generated a key concept in developmental biology. Int. J. Dev. Biol. 45, 1–11 (2001) PubMed Gilbert, S. F. Developmental Biology 7th edn: 317–321 (2004) FREE

1 July 2004 | doi:10.1038/nrn1450 Taking a leaf from the book of cell fate Natalie DeWitt, Senior Editor, Nature "Oh God! That one might read the book of fate!" Henry IV, William Shakespeare With all due respect to Shakespeare, let us consider another rich, elegant, and infinitely more knowable book of fate than the one King Henry IV lamented. This book describes the journey of a fertilized egg as it metamorphoses into an embryo, a fetus, a juvenile, and finally an adult. Fate maps are the constellations by Quail cells (black) 24 hours after transplantation into the chick which developmental biologists neural plate. navigate the journey of development, Image first published in Nature and thanks to pioneering work of early Reviews Neuroscience 2, 763–771 embryologists, this book of fate can be (2001) found, at least in rudimentary form, on library bookshelves. The earliest fate maps — of invertebrate sea creatures for the most part — date back to the 1880s. In 1905, Edwin Conklin published a remarkably comprehensive and insightful collection of ascidian fate maps, presciently observing that substances are segregated to particular cells during development and that this distribution is correlated with the fate of those cells. However, it was probably Walter Vogt who paved the way for modern vertebrate fate-mapping techniques. In 1929, Vogt developed a method of marking groups of cells on the surface of amphibian embryos with dyes, then following the movement of those cells throughout gastrulation. His fate maps have been confirmed and refined by modern microscopy and dye-injection techniques. Variations in the fate maps of different amphibian species were uncovered, and lineage-marking studies by Smith and Malcinski and Lundmark (combined with scanning electon microscopy in Lundmark's case) showed that this could be attributed to variations in the gastrulation mechanism between different species. Amphibian fate maps continue to inform the design and interpretation of modern-day experiments aimed at understanding the molecular mechanisms that regulate developmental movements. Fate maps relying on vital dyes and radioactive markers were to some extent confounded by the tendency of the marking substances to diffuse, greatly hampering the resolution of the techniques. A milestone was reached in 1969, when Nicole Le Douarin pioneered the use of chick–quail chimaeras for fate mapping. Her experiments are notable for their elegance, the cellular resolution they provided, and the foundation they built for future studies of neural-crest migration, epithelial–mesenchymal interactions and neural-plate formation. Her studies exploited two characteristics of quails and chickens: first, embryos of the two species develop in similar ways, and second, quail and chicken cells can be distinguished from each other using a simple DNA staining technique. Le Douarin discovered that if she transplanted bits of the quail embryo into the corresponding region of the chick embryo while it was still in the egg, the transplanted tissue would meld perfectly to the host tissue, and normal development would ensue, even for some time after birth. She also noticed that during interphase, quail cells possess a dense patch of nucleolar heterochromatin when stained with Schiff's k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730663679? reagent. By contrast, chick heterochromatin is diffuse and the nucleoli are indistinct. Recognizing that this distinction provided a way of 'marking' the cells, Le Douarin went on to show that quail and chick cells can be distinguished in multiple organ systems, and throughout development to adulthood. Le Douarin used the staining technique to follow the fate of the transplanted cells during development. In one proof-of-principle experiment, she showed that chimaeric birds can be used to map the fate of neural-crest cells, in what has since become a classic approach for studying this migratory population of embryonic cells. She excised a region of an early embryonic chick neural tube and replaced it with the corresponding region of a quail embryo. After 6 days of development, she sectioned the host embryo and found that the quail neural crest cells had contributed to various tissues, such as chromaffin and pigment cells, and ganglion cells of the peripheral nervous system. She went on show that the chick–quail chimaeras could be useful for studying epithelial– mesenchymal interactions during lung development, and the differentiation of glycogen-producing hepatic cells during liver development. Fate-mapping techniques have continued to evolve, increasing in their sensitivity, level of resolution and ability to image in real time. Le Douarin could only trace the fate of groups of cells, but modern investigators can mark single cells using retroviruses, fluorescent dyes or genetic markers, and can combine fate mapping with ablation of genes and cells. And now, thanks to the development of a new generation of markers and microscopes, even fate mapping's holy grail — observing the fate of individual cells in real time in living organisms — has become reality.

REFERENCES ORIGINAL RESEARCH PAPERS Vogt, W. Gestaltungsanalyse am Amphibienkeim mit ortlicher Vitalfarbung. II. Teil Gastrulation und Mesodermbildung bie Urodelen und Anuren. Wilhelm Roux Arch. Entwicklungsmech. Org. 120, 384–706 (1929) Le Douarin, N. M. Particularites du noyau interphasique chez la Caille Japonaise (Coturnix Coturnix Japonica). Bull. Biol. Fr. Belg. 103, 435–452 (1969) PubMed

FURTHER READING Conklin, E. G. The organization and cell lineage of the ascidian egg. J. Acad. Nat. Sci. Phil. 12, 1–119 (1905) Smith, J. C. & Malacinski, G. M. The origin of the mesoderm in an anuran, Xenopus laevis, and a urodele, Ambystoma mexicanum. Dev. Biol. 98, 250–254 (1983) PubMed Lundmark, C. Role of bilateral zones of ingressing superficial cells during gastrulation of Ambystoma mexicanum. J. Embryol. Exp. Morphol. 97, 47–62 (1986) PubMed Clarke, J. D. W. & Tickle, C. Fate maps old and new. Nature Cell Biol. 1, E103–E109 (1999) Article PubMed Schoenwolf, G. C. Cutting, pasting and painting: experimental embryology and neural development. Nature Rev. Neurosci. 2, 763–771 (2001) Article Gilbert, S. F. Developmental Biology 7th edn: 308–309; 348–351 (2004) FREE

1 July 2004 | doi:10.1038/nrn1451 Inhibit thy neighbour Katrin Bussell, Senior Editor, Nature Reviews Molecular Cell Biology How does a homogeneous population of neurogenic ectodermal cells give rise to a specific pattern of neuronal precursor cells or neuroblasts? We now know that just one cell within a group adopts a different fate and then sends out inhibitory messages, using the Notch–Delta pathway, to stop its neighbours taking on the same fate, but how did we arrive at this understanding? Back in 1937, Donald F. Poulson was studying embryonic development in Drosophila melanogaster by using deficiencies involving the entire X chromosome or reasonably large portions thereof. The results of one such deficiency, known as Notch-8, were detailed. “The most striking feature of such eggs is that they contain very little or no endoderm or mesoderm ... the process of germ layer formation has been interfered with seriously... The ectoderm proliferates especially along the ventral mid-line and produces what appears to be a semblance of the early nervous system.” It was almost 50 years before Chris Doe and Corey Goodman, studying the neural ectoderm of grasshoppers, suggested a mechanism that we now know as lateral inhibition. Using laser microbeams to ablate one or more ectodermal cells in a group, they found that neuroblasts are specified by cell interactions. Initially, each undifferentiated cell within a sheet of neural ectodermal cells has an equal chance of becoming a neuroblast, but only one cell within a group takes on this role. Interactions between the cells of a group allow this one cell to enlarge into the neuroblast, which somehow prevents its neighbouring cells from taking on the same identity; these cells instead become support cells or die. So, cell–cell interactions were obviously important, but what molecules were behind the mechanism? In 1985, Spyros Artavanis-Tsakonas' group used chromosomal walking and the Sanger sequencing method (outlined only a few years earlier) to determine the nucleotide sequence of four overlapping cDNA clones encompassing the Notch locus. From this, the predicted 2,703-amino-acid sequence was determined. About half of the predicted polypeptide sequence contains 36 tandem cysteine-rich repeats of ∼40 amino acids, with 6 cysteines positioned at specific intervals within each repeat. Searching the Protein Information Resource database identified considerable homology of these repeats with epidermal growth factor (EGF). At the time, the functional significance of this homology was unclear, but Artavanis-Tsakonas and colleagues later showed that these are important in the interaction between Notch and its ligands. Indeed, hydropathy plots by Artavanis-Tsakonas' group implied that Notch spanned the membrane, and the EGF repeats would therefore be extracellular. The authors discussed, on the basis of genetic studies, the possibility that the products of Notch, Delta and Enhancer of split — a known target of Notch signalling — might interact. They proposed that “Notch and all or some of the other neurogenic loci may be involved in a cellular mechanism that mediates and interprets cell–cell interactions, which result in the correct differentiation of the neurogenic region” — a hypothesis that turned out to be correct. We now know that Notch and Delta are the defining players in the signalling pathway that underlies lateral inhibition. However, there is still much to learn about this process — several other molecules also have key roles, and the mechanism by which Notch–Delta interactions activate gene transcription is still incompletely understood. REFERENCES

ORIGINAL RESEARCH PAPERS Poulson, D. F. Chromosomal deficiencies and embryonic development of Drosophila melanogaster. Proc. Natl Acad. Sci. USA 23, 133–137 (1937) Doe, C. Q. & Goodman, C. S. Early events in insect neurogenesis. II. The role of cell interactions and cell lineage in the determination of neuronal precursor cells. Dev. Biol. 111, 206–219 (1985) PubMed Wharton, K. A. et al. Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43, 567–581 (1985) Article PubMed

FURTHER READING Poulson, D. F. The effects of certain X-chromosome deficiencies in the embryonic development of Drosophila melanogaster. J. Exp. Zool. 83, 271–325 (1940) Poulson, D. F. in Biology of Drosophila (ed. M. Demerec) 168–274 (1950) Poulson, D. F. in D. Mel. D. I. S. 42, 81 (1967) Fehon, R. G. et al. Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61, 523–534 (1990) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 166–168 (2004) FREE

Milestone 4 (1952)

1 July 2004 | doi:10.1038/nrn1452 Symmetry breaking and computer simulation Christopher Surridge, Senior Editor, Nature Alan Turing's 1952 paper 'The Chemical Basis of Morphogenesis' — his last before committing suicide in 1954 — might not appear on everyone's list of Milestones as it fails to answer any specific question in developmental biology. However, by showing that patterns could be generated by simple chemical reactions, together with diffusion, it marked a change in how the processes of development were viewed. It also contains the first applications of computer modelling in biology. Turing attempted to discover how the symmetry of a homogeneous mass of identical cells could be broken to form a particular pattern of two or more spatially distinct cell types. He saw that, if the differentiated state was more thermodynamically stable than the homogeneous state, then any small deviations from complete homogeneity, such as were produced by the stochastic fluctuations that occur all the time, would be amplified and homogeneity would be lost. To set up this kind of instability, Turing investigated a hypothetical situation in which cells produced two or three different 'morphogens' — freely- diffusing molecules that controlled their own production and the cells' subsequent development. In the two-morphogen case, the first diffused slowly and enhanced morphogen production, and the second diffused more quickly and was inhibitory. This scheme could be applied to a ring of cells using “relatively elementary mathematics”. Turing also had, at his disposal, Manchester University's newly installed Ferranti Mark I, the first ever commercially available computer. With this he showed that depending on the values assigned to the model's parameters, six different classes of self-organizing pattern could result, some reminiscent of biological structures. The most evocative had standing waves of morphogen around the ring, reminding Turing of the distribution of tentacles around a hydra or the distribution of petals in a flower. By considering a sphere of cells, Turing also modelled the blastula stage of embryo development, a point at which some embryos begin patterning. Here, the equations predicted a single, randomly positioned area of maximum morphogen concentration, which could be identified with the vegetal pole formed by amphibian and sea-urchin embryos. Although no event in embryogenesis has been found that relies on the exact scheme that Turing outlined, reaction–diffusion models of the Turing type have been widely explored. Most notably, Alfred Gierer and Hans Meinhardt showed that the more general case of local self-enhancement coupled with longer-range inhibition was not only sufficient but, in fact, necessary for pattern formation to occur. Many processes in both animals and plants depend on such Turing–Gierer–Meinhardt mechanisms. That is not to say that Turing's original proposal wouldn't work. In the early 1990s, purely chemical systems producing Turing patterns were developed and 5 years later, a natural Turing pattern was found on the skin of an angelfish. However, the significance of Turing's paper is not in what it says — although anyone trying to present highly mathematical arguments to a non-mathematical audience should read it as an object lesson on how it should be done — but rather for its demonstration that development could be approached in a quantitatively rigorous fashion. REFERENCES

ORIGINAL RESEARCH PAPERS Turing, A. M. The chemical basis of morphogenesis. Phil. Trans. R. Soc. Lond. B 327, 37–72 (1952) Gierer, A. & Meinhardt, H. A. A theory of biological pattern formation. Kybernetik 12, 30–39 (1972) PubMed Castets, V. et al. Experimental evidence of a sustained standing Turing-type nonequilibrium chemical pattern. Phys. Rev. Lett. 64, 2953–2965 (1990) Article PubMed Ouyang, Q. & Swinney, H. L. Transition from a uniform state to hexagonal and striped Turing patterns. Nature 352, 610–612 (1991) Article Kondo, S. & Asai, R. A reaction-diffusion wave on the skin of the marine angelfish Pomacanthus. Nature 376, 765–768 (1995) Article

FURTHER READING Meinhardt, H. Different strategies for midline formation in bilaterians. Nature Rev. Neurosci. 5, 502–510 (2004) Article Gilbert, S. F. Developmental Biology 7th edn: 18–21 (2004) FREE

Milestone 5 (1952)

1 July 2004 | doi:10.1038/nrn1453

Turning back time Nick Campbell, Associate Editor, Nature Reviews Genetics Dolly the sheep — that cuddly media darling of the late 1990s — actually came 35 years after what was perhaps an even more significant cloning breakthrough: a much less-feted 1960s amphibian, that might have been dubbed Molly the frog had the popular press been on the case back then. The legacy of Dolly and Molly is that we now know that it is possible to turn back developmental time and make a complete organism from a nucleus taken from an adult cell. It is difficult to imagine breakthroughs in developmental research that could have greater social and medical repercussions in future than these. The groundwork for these crucial breakthroughs was laid more than 50 years ago. In 1952, Robert Briggs and Thomas King, working on a frog, Rana pipiens, became the first to successfully transplant living nuclei in multicellular organisms. They transplanted blastula nuclei into enucleated eggs, which then developed into normal embryos. This was a huge technical breakthrough and provided a spur for those interested in finding out exactly what caused the changes seen during cell differentiation. However, the successful transplants that Briggs and King performed were of undifferentiated nuclei. Until it was possible to accomplish the same feat with a differentiated nucleus, it would remain an open question as to whether the genome itself somehow changed during development or whether it was the way genes were expressed that was responsible for differentiation. It was John Gurdon's work on Xenopus laevis in the late 1950s and early 1960s, culminating in his seminal 1962 paper, that resolved this pitech.com ). A valid user ID conundrum: genes were not lost or changed during cell differentiation — they were just differentially expressed. In his landmark study, Gurdon transplanted intestinal epithelium-cell nuclei from Xenopus tadpoles into dministrator enucleated frog eggs and managed to produce 10 normal tadpoles: Molly and her fellow clones. The logical consequence of Gurdon's success — that the nuclei of differentiated cells retain their totipotency — provided a key conceptual advance in developmental biology. However, Gurdon didn't immediately receive the universal acclaim he deserved. The problem was that Briggs and King's earlier work on Rana pipiens indicated that nuclear transfer might not be able to reverse embryonic stem-cell differentiation, so many were sceptical about Gurdon's success in Xenopus. However, Gurdon's subsequent production of normal fertile adult animals from eggs with genetically marked nuclei transplanted from differentiated tadpole cells silenced the detractors. It proved once-and- for-all that the genome remained intact during differentiation and that the epigenetic changes to the somatic-cell nucleus were reversible.

4/172.24.132.133/http://ad.doubleclick.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730681310? It was more than three decades before a breakthrough of a similar magnitude to Gurdon's was to come. The year before their landmark 1997 publication, Ian Wilmut and his team managed to clone a sheep from an embryo-derived cell line that they induced to be quiescent. However, the real breakthrough came when they successfully applied a similar strategy to produce a normal sheep cloned from adult cells. The scientific and social implications of this success were huge. Although Gurdon's seminal amphibian work had shown that the nuclei from differentiated cells could have their developmental clock turned back, Dolly was the first clone to be produced from an adult cell. Coupled with the fact that this success came in a mammal, the possibility that humans could be cloned from adult cells was an obvious one and saturation media coverage was the result. However, the scientific legacy of Briggs, King, Gurdon and Wilmut is a much broader one: the knowledge that differentiation of a cell is not irreversible and that nuclei can be reprogrammed has driven expansion of such fields as stem-cell biology and epigenetics. Moreover, should cloning fulfill its enormous therapeutic potential, the debt the world owes these pioneers will be equally large.

REFERENCES ORIGINAL RESEARCH PAPERS Briggs, R. & King, T. J. Transplantation of living nuclei from blastula cells into enucleated frogs' eggs. Proc. Natl Acad. Sci. USA 38, 455–463 (1952) Gurdon, J. B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 34, 93–112 (1962) Wilmut, I. et al. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997) Article PubMed

FURTHER READING Rhind, S. M. et al. Human cloning: can it be made safe? Nature Rev. Genet. 4, 855–864 (2003) Article PubMed Solter, D. Mammalian cloning: advances and limitations. Nature Rev. Genet. 1, 199–207 (2000) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 85–90 (2004) FREE

WEB SITES John Gurdon's laboratory: http://www.welc.cam.ac.uk/groups/gurdon.html The Roslin Institute: http://www.roslin.ac.uk/

Milestone 6 (1957)

1 July 2004 | doi:10.1038/nrn1454 Out on a limb Heather Wood, Senior Editor, Nature Reviews Neuroscience If babies could talk, they would no doubt testify that hands and feet are fascinating things. If they were in a particularly philosophical mood, they might — like many developmental biologists — wonder why there are five fingers on each hand, and how the parts of the limb are so neatly patterned and articulated. Limb development is a key research topic, which encompasses issues of cell differentiation, tissue patterning and signalling. We owe much of our current understanding of limb morphogenesis to John Saunders and his colleagues, whose pioneering experiments in chick embryos led to the discovery of key signalling centres in the limb bud. In 1948, Saunders had shown that removal of a thickened layer of ectoderm that rims the distal tip of the limb bud — dubbed the apical ectodermal ridge (AER) — causes truncation of the limb. In a 1957 paper, Saunders, along with John Cairns and Mary Gasseling, grafted prospective thigh mesoderm from the chick leg bud to the apex of the wing bud, and they found that this mesoderm generated foot structures if it was placed in contact with the AER. However, if wing mesoderm was allowed to ingress between the grafted tissue and the AER, the grafted mesoderm retained its thigh identity. Experiments such as these led to the suggestion that the AER is not only required for limb outgrowth, but it also provides signals that allow specific structures to form at different proximo–distal levels of the limb axis. Gail Martin and colleagues subsequently attributed the signalling activity of the AER to molecules of the fibroblast growth factor (FGF) family. It has become accepted that signals from the AER maintain a population of undifferentiated cells that give rise to successive skeletal elements of the proximo–distal limb pattern. However, recent findings from the laboratory of Clifford Tabin indicate that this pattern is not established progressively during limb-bud development, but instead results from the expansion of cell populations that are specified in the young limb bud. Saunders was also interested in how the antero–posterior pattern of digits is generated in the limb. In 1968, Saunders and Gasseling identified a region at the posterior margin of the wing bud — later termed the zone of polarizing activity (ZPA) — which, when transplanted to an anterior position in the wing-bud margin, caused a mirror-image duplication of digits. In 1975, Cheryl Tickle, Dennis Summerbell and Lewis Wolpert proposed that the ZPA releases a diffusible morphogen, establishing a gradient such that the posterior-most digit arises closest to the source of the morphogen, and the more anterior digits emerge at sites with progressively lower morphogen concentrations. As retinoic acid (RA) can mimic the polarizing effect of the ZPA, it was initially thought that this might be the morphogen. However, Tabin's group showed that the ZPA signal is Sonic hedgehog, and that RA primarily induces the formation of an ectopic ZPA. The concept of inductive interaction in morphogenesis that was formulated by Saunders et al. has proved to be remarkably robust, and it k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730686254? represents a fundamental model to elucidate the importance of signalling activity and tissue interactions for the development of complex structures.

REFERENCES ORIGINAL RESEARCH PAPERS Saunders, J. W. The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 129, 5–44 (1948) Saunders, J. W. et al. The role of the apical ridge of ectoderm in the differentiation of the morphological structure and inductive specificity of limb parts in the chick embryo. J. Morphol. 101, 57–87 (1957) Saunders, J. W. & Gasseling, M. in Epithelial–Mesenchymal Interactions (eds Fleischmayer, R. & Billingham, R. E.) 78–97 (Williams and Wilkins, Baltimore, 1968).

FURTHER READING Tickle, C. et al. Positional signalling and specification of digits in chick limb morphogenesis. Nature 254, 199–202 (1975) PubMed Tickle, C. et al. Local application of retinoic acid to the limb bond mimics the action of the polarizing region. Nature 296, 564–566 (1982) PubMed Riddle, R. D. et al. Sonic-hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401– 1416 (1993) PubMed Martin, G. R. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571–1586 (1998) PubMed Dudley, A. T. et al. A re-examination of proximodistal patterning during vertebrate limb development. Nature 418, 539–544 (2002) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 534–540 (2004) FREE

Milestone 7 (1963)

1 July 2004 | doi:10.1038/nrn1455 Common sense Jon Reynolds, Assistant Editor, Nature Cell Biology Formation of the mature nervous system requires axons to navigate to their correct targets and establish synaptic connections. Growing axons produce highly motile structures, called growth cones, which guide the axon to its target. They do this by responding to specific guidance molecules that either attract or repel the growth cone. The idea that axons are guided principally by molecular determinants, rather than mechanical determinants such as cells, extracellular material and other neurons, was established by Roger Sperry in 1963. However, it was not until more recent times — through the discovery of guidance molecules such as netrins, semaphorins, ephrins and Slits — that Sperry's chemoaffinity hypothesis became widely accepted as a common mechanism for guidance of not just axons, but of all cells. In 1981, Sperry received the Nobel Prize for Physiology or Medicine “for his discoveries concerning the functional specialization of the cerebral hemispheres”. He studied patients with epilepsy in whom the corpus callosum — the bundle of axons fibres that connects the two brain hemispheres — had been severed to prevent seizures. A number of tests revealed how the two brain hemispheres hold independent streams of conscious awareness, perceptions, thoughts and memories and, importantly, that neuronal connections are formed and maintained with a high degree of precision. Having demonstrated that the establishment of specific neuronal connections is fundamental to function, Sperry turned to look at how connections are made, and used his chemoaffinity hypothesis to explain how axons find the correct target. Others had raised the possibility that chemical determinants might function in axon guidance, but it was Sperry that provided the direct histological evidence and proposed the chemoaffinity hypothesis for axon guidance. In a series of elegant experiments using the retinotectal system of the newt, he sectioned the optic nerves and rotated the eyes 180 degrees. He wanted to know whether vision would be normal following regeneration or whether the animal would forever view the world 'upside down'. If the latter held true, this would show that the nerves were somehow guided back to their original sites of termination; however, restoration of normal vision would mean that the nerves had terminated at new sites. Sperry showed that these animals did indeed view the world 'upside down'. It was these studies that led Sperry to propose that “intricate chemical codes under genetic control” guide axons to their targets — his chemoaffinity hypothesis. In his original hypothesis, Sperry proposed that different cells bear distinct cell-surface proteins that serve as markers — an idea that required an unsatisfactorily large number of proteins. He subsequently revised his model to suggest that dual gradients of guidance cues in the afferent and target fields would allow correct axon targeting. There is now extensive experimental data to support the chemoaffinity hypothesis, and the requirement for gradients of receptor and/or ligand (such as ephrins and Eph receptors) in both the projection and target regions is well established. Although certain aspects and details of Sperry's model are unproven or incorrect, the basic notion of the chemoaffinity hypothesis has now become dogma in developmental neurobiology.

REFERENCES ORIGINAL RESEARCH PAPER Sperry, R. W. Chemaffinity in the orderly growth of nerve fiber patterns and connections. Proc. Natl Acad. Sci. USA 50, 703–710 (1963) PubMed

FURTHER READING Meyer, R. L. Roger Sperry and his chemoaffinity hypothesis. Neuropsychologia 36, 957–980 (1998) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 444–458 (2004) FREE

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730690452? PUBLI

Milestone 8 (1969)

1 July 2004 | doi:10.1038/nrn1456 Shaping destiny Amanda Tromans, Senior Editor (News and Views), Nature The idea of one group of cells telling another what type of tissue it should become — a phenomenon known as induction — is a familiar theme in embryonic development. One of the earliest instances of this process is the formation of mesoderm, which, along with ectoderm and endoderm, is one of the three main tissue layers in early embryos. The discovery that mesoderm is induced from ectoderm, under instructions from endoderm, dates back to a seminal publication in 1969. It would be another 20 years, however, before researchers would understand what form those instructions take. The amphibian embryo is a popular model organism for developmental biology research. At early (blastula) stages, the embryo consists of two relatively easily distinguishable parts — the 'animal' and 'vegetal' hemispheres. When excised and allowed to develop alone, the animal part develops into ectodermal tissues, whereas the vegetal half forms endodermis. But how does mesoderm form? P. D. Nieuwkoop tackled this problem through a series of meticulous dissection experiments. By excising different portions of Ambystoma mexicanum (axolotl) blastulae, and culturing them alone or in combination with other portions, he concluded that the mesoderm develops from the ectodermal (animal) part of the embryo, but requires contact with the endodermal (vegetal) part to do so. The implication was that signals released from the vegetal half instruct the animal half. Over the years, some attempts were made to purify the factors responsible, from embryos and from animal tissues, but without much success. Then, in 1987, J. M. W. Slack and colleagues published the results of testing various different growth factors on ectoderm explants from Xenopus blastulae. They found that, at low concentrations, basic fibroblast growth factor (bFGF) could induce mesoderm, as judged by histological criteria. Crucially, they also showed that when animal and vegetal explants were cultured together, the addition of heparin could prevent mesoderm induction — suggesting that the natural mesoderm- inducing signal from the vegetal half is a molecule that can be inhibited by heparin. FGF is one such molecule. Later that year, David Kimelman and Marc Kirschner published the results of similar experiments. These authors used a different indicator of mesoderm induction — the levels of mRNA encoding cardiac actin — but they, too, found that bFGF can induce mesoderm. They also discovered that transforming growth factor-β (TGF-β) is required to boost cardiac actin expression to the level seen normally in embryos. Moreover, they found that FGF mRNA is present in early embryos. Three years later, J. C. Smith et al. discovered that in mammals, a likely mesoderm inducer is activin A — a protein that was best known until then for its roles in adult organisms, butwhich was now shown to have a similar sequence and activity to a member of the Xenopus TGF-β family that can induce mesoderm.

REFERENCES ORIGINAL RESEARCH PAPERS Nieuwkoop, P. D. The formation of the mesoderm in urodelean amphibians. I. Induction by the endoderm. Wilhem. Roux Archiv. 162, 341–373 (1969) Slack, J. M. W. et al. Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature 326, 197–200 (1987) Article PubMed Kimelman, D. & Kirschner, M. Synergistic induction of mesoderm by FGF and TGF-β and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51, 869–877 (1987) Article PubMed Smith, J. C. et al. Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A. Nature 435, 729–731 (1990) Article

FURTHER READING Weeks, D. L. & Melton, D. A. A maternal mRNA localized to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-β. Cell 51, 861–867 (1987) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 321–322 (2004) FREE

.doubleclick.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730694295? Milestone 9 (1971)

1 July 2004 | doi:10.1038/nrn1457 Reinventing reproduction Emma Green, Copy Editor, Nature Reviews Neuroscience In 1959, the first repeatable procedure for fertilizing mammalian ova in vitro was reported in Nature by M. C. Chang. This marked the beginning of assisted reproductive technology and would lead to the development of what are now common practices for helping couples who have difficulties in conceiving naturally. Chang used capacitated rabbit spermatozoa, collected from the uterus of a mated rabbit in oestrous, and unfertilized ova, collected from rabbits that had been induced to ovulate by the injection of sheep pituitary extract. After culturing ova and spermatozoa together at 38°C for 3–4 hours, ova were transferred to flasks that contained heated (55°C) rabbit serum for ∼18 hours. Fertilized and cleaved ova (36 out of 266) were then transplanted into six rabbits that had been injected with pituitary extract 18 hours previously. Of the 6 recipients, 4 delivered 15 healthy young between them. Over the next decade, work began on the application of Chang's technique in humans. In 1971, Edwards and colleagues reported the in vitro growth of human oocytes to the blastocyst stage. Previous attempts in mammals had led to embryos that were incapable of sustained growth. The difference in this technique compared with previous attempts was the use of preovulatory oocytes (immature ova) rather than ova collected after ovulation. The method resulted in the birth of the first in vitro fertilized child in 1978 using the natural ovulating cycle (see Steptoe and Edwards, 1978). It also led to the introduction of fertility drugs to boost the collection of oocytes and control the time of ovulation (see Trounson et al., 1981), and to freeze the excess embryos that were created (see Trounson and Mohr, 1983). In 1987, Laws-King et al. reported a technique that would revolutionize assisted reproductive technology and offer hope to couples where other infertility treatments had failed. The technique — referred to as SUZI (sub- zonal insertion)— involved the microinjection of sperm under the zona pellucida of human oocytes. Using preovulatory oocytes and spermatozoa that had undergone capacitation through chemical exposure, a single spermatozoon was microinjected into the perivitelline space. Five out of seven oocytes fertilized, three went on to cleave and one reached the six- cell stage of cleavage. The SUZI technique had profound implications for the treatment of severe male infertility, and offered hope to men with completely immotile, immature or abnormal spermatozoa. However, there were drawbacks. When the technique was used for treating infertility, multiple sperm were injected under the zona pellucida to increase the chance of fertilization. This increased the risk of polyspermy, a lethal condition when more than one sperm enters the oocyte. This problem was overcome in 1992, with the report by Palermo and co- workers of successful human pregnancies in three out of four couples that had previously been unsuccessful with SUZI and other in vitro techniques. Successful fertilization using the ICSI (intracytoplasmic sperm injection) k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730698170? method required the microinjection of just a single spermatozoon into the ooplasm of an oocyte, thereby bypassing spermatozoa binding and penetration into the zona pellucida and the fusion of sperm by the acrosome reaction. Today, ICSI is commonly used in the treatment of infertility. However, as with most manipulations of nature, there are associated risks; for example, the possibility of inheriting certain genetic conditions might be greater. As technology and medicine evolves, these risks will be measured and hopefully minimized. REFERENCES

ORIGINAL RESEARCH PAPERS Chang, M. C. Fertilization of rabbit ova in vitro. Nature 184, 466–467 (1959) PubMed Steptoe, P. C. et al. Human blastocysts grown in culture. Nature 229, 132–133 (1971) PubMed Laws-King, A. et al. Fertilization of human oocytes by microinjection of a single spermatozoon under the zona pellucida. Fertil. Steril. 48, 637–642 (1987) PubMed Palermo, G. et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 340, 17–18 (1992) Article PubMed

FURTHER READING Steptoe, P. C. & Edwards, R. G. Birth after the reimplantation of a human embryo. Lancet 2, 366 (1978) Article PubMed Trounson, A. O. et al. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science 212, 681–682 (1981) Trounson, A. & Mohr, L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305, 707–709 (1983) PubMed Gilbert, S. F. Developmental Biology 7th edn: 683–686 (2004) FREE

1 July 2004 | doi:10.1038/nrn1458 Systems biology ahead of its time Magdalena Skipper, Editor, Nature Reviews Genetics In this genomic age, we take for granted the availability of complete genome sequences, proteome sets or even interactomes — exhaustive descriptions of protein–protein interactions. Instead of focusing on an individual gene or an individual problem, the challenge is to take a 'holistic approach' to biology, in what has been dubbed 'systems biology'. Those who think that systems biology was only born with the advent of whole-genome sequencing, however, should consider the tour-de-force efforts of John Sulston and colleagues, who in the late 1970s and early 1980s set out to determine the entire cell lineage of C. elegans. The work on the worm had something 'systematic' about it from the beginning, as exemplified by its conscious choice as a model — for neurobiology, in particular — by Sydney Brenner. Following the classical observations that nematode early development was invariant, Sulston and Horvitz set out to map out all of these cell divisions. They, aided by Judith Kimble, achieved this bold undertaking by directly watching cell divisions, migrations and death in living nematodes, under Nomarski Image courtesy of S. optics. Reichett, MRC Laboratory of Molecular Biology, Cambridge, UK. Detailed lineage information had a huge impact on subsequent research — for example, Sulston and Horvitz saw that in a number of lineages some cells always died in an invariant way. Focusing on the fate of these cells in subsequent mutant screens allowed researchers (mainly the Horvitz group) to dissect the genetic pathway for programmed cell death (see Milestone 15). Defined neuronal cell lineages had a similar impact on neurobiology studies in the worm. Six years after the post-embryonic lineage was defined, Sulston and colleagues turned to the embryo to show the divisions involved in transforming a zygote into the 671-celled embryo at hatching. Importantly, the authors described in detail the early asymmetric cell divisions and the developmental potential of what they called the founder cells. Subsequent work from many labs showed how these asymmetric cell divisions are achieved and that this asymmetry is important for the lineage specification and normal development. So, should we push back the birth date of systems biology by some 20 years? It might seem fitting to think of John Sulston as a pioneer of this approach. After all, he was to go on to be one of the early champions of the Human Genome Project and subsequently one of the leaders of the publicly-funded international consortium.

REFERENCES ORIGINAL RESEARCH PAPERS Sulston, J. E. & Horvitz, H. R. Post-embryonic cell lineage of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–156 (1977) PubMed Sulston, J. E. et al. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983) PubMed k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730703721? FURTHER READING Gilbert, S. F. Developmental Biology 7th edn: 251–253 (2004) FREE

1 July 2004 | doi:10.1038/nrn1459 Order: it's in the genes Tanita Casci, Senior Editor, Nature Reviews Genetics Thirty years ago, men were men and flies were flies and the two species were thought to share no more than a handful of genes and a partiality to bananas. Then came the discovery that so-called 'homeotic genes' controlled axial patterning, and that their sequence and function was conserved between distant organisms. The unexpected realization that vertebrates and invertebrates were evolutionarily related through such an essential developmental process altered the course of developmental genetics — it turned flies into authorized models for animal development and kickstarted the field of vertebrate molecular embryology. It was Bateson who got the ball rolling. In 1894, he described natural variants in which body segments had acquired the identity of other regions. The phenomenon, which he called 'homeosis', was shown by T. H. Morgan to be heritable in flies. But it wasn't until Ed Lewis put his mind to it 50 years later that the underlying genetics began to be unravelled. By examining the genotype–phenotype relationship of homeotic mutations at the fly bithorax cluster of genes (BX- C) — for example, those that transformed the third thoracic segment into the second, giving the flies two sets of wings — he reported that the BX-C consisted of distinct genetic elements and that mutations map in an order that corresponds to the anterior–posterior (A–P) order of the body parts that they affect. This feature, called spatial collinearity, would turn out to be a defining feature of both vertebrate and invertebrate homeotic genes. Lewis showed remarkable vision by arguing that the identity of an individual body segment is produced by the particular combination of BX-C genes, and that these were activated in reponse to an A–P gradient. On the molecular biology front, techniques were speeding up. Two teams, one led by Matthew Scott and the other including William McGinnis, Michael Levine and Walther Gehring, showed in 1984 that homeotic genes share a 184-bp sequence called the 'homeobox'. This is translated into a 60-amino-acid DNA-binding motif — the homeodomain — by which homeobox (or Hox) genes bind to DNA and control transcription. It was only a short time before the homeobox was used in homology searches to pull out more homeotic genes, in flies and farther afield: frogs, chickens, zebrafish, mice and humans all had Hox genes that, like those of flies, were arranged in clusters. In 1989, Duboule and Dollé and the Krumlauf laboratory showed by in situ hydridization that mouse and fly Hox clusters showed a similar spatial and functional organization. Not only did genes in vertebrate and invertebrate clusters have spatial collinearity, but they were expressed in a temporal order that matched their physical order on the chromosome (that is, they showed 'temporal collinearity'). In addition, fly Hox genes — which map to two adjacent clusters, BX-C and ANT-C — could, by and large, be matched up with genes at similar positions on each of the four paralogous vertebrate clusters. This was a remarkable breakthrough and a vindication of Lewis' belief that all homeotic genes are evolutionarily related. Even before the publication of Lewis' Nobel-prize winning paper, homeotic phenotypes had led to some surprisingly accurate predictions about how homeotic 'selector genes', which Garcia-Bellido insightfully named and described, might specify the development of groups of cells. Three k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730707740? decades on, we have an astonishing amount of information on the genes that pattern the axes and segments of an animal — although precisely how Hox genes combine to produce the numerous, yet stereotypical, morphological patterns that we see still escapes us. REFERENCES ORIGINAL RESEARCH PAPERS Garcia-Bellido, A. Genetic control of wing disc development in Drosophila. Ciba Found. Symp. 29, 161–182 (1975) PubMed Lewis, E. A gene complex controlling segmentation in Drosophila. Nature 276, 565–570 (1978) PubMed McGinnis, W. et al. A conserved sequence in homeotic genes of the Drosophila Antennapedia and bithorax complexes. Nature 308, 428–433 (1984) PubMed Scott, M. P. & Weiner, A. J. Structural relationships among genes that control development: sequence homology between the Antennapedia, Ultrabithorax, and fushi tarazu loci of Drosophila. Proc. Natl Acad. Sci. USA 81, 4115–4119 (1984) PubMed Laughon, A. & Scott, M. P. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins. Nature 310, 25–31 (1984) PubMed Duboule, D. & Dollé, P. The structural and functional organization of the murine Hox gene family resembles that of Drosophila homeotic genes. EMBO J. 8, 1497–1505 (1989) PubMed Graham, A. et al. The murine and Drosophila homeobox gene complexes have common features of organization and expression. Cell 57, 367–378 (1989) Article PubMed

FURTHER READING Garcia-Bellido, A. et al. Developmental compartmentalisation of the wing disc of Drosophila. Nature New Biol. 245, 251–253 (1973) PubMed Hafen, E. et al. Regulation of Antennapedia transcript distribution by the bithorax complex in Drosophila. Nature 307, 287–289 (1984) Carrasco, A. E. et al. Cloning of an X. laevis gene expressed during early embryogenesis coding for a peptide region homologous to Drosophila homeotic genes. Cell 37, 409–414 (1984) Article PubMed Sanchez-Herrero, E. et al. Genetic organization of Drosophila bithorax complex. Nature 313, 108–113 (1985) PubMed Patel, N. H. et al. Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58, 955–968 (1989) González-Reyes, A. & Morata, G. The developmental effect of overexpressing a Ubx product in Drosophila embryos is dependent on its interactions with other homeotic products. Cell 61, 515–522 (1990) Article PubMed Holland, P. W. H. in Milestones in Systematics: Systematics Association Special Volume Series 67 (eds Williams, D. M. & Forey, P. L.) 261–275 (Boca Raton, Florida, CRC Press; in the press). Gilbert, S. F. Developmental Biology 7th edn: 377–381 | 754–761 (2004) FREE

Milestone 12 (1980s)

1 July 2004 | doi:10.1038/nrn1460

Tools of the trade Alison Schuldt, Senior Editor, Nature Cell Biology Imagine trying to get to the lab first thing on a Monday morning without your alarm clock and hot shower. It is all too easy to take the technical achievements around us for granted, although we would quickly be stopped in our tracks without them. The same is true in research: perhaps only those who have been in the field long enough can truly appreciate the importance of methods that overcame technical hurdles. In developmental biology, one of the most significant examples has been the ability to manipulate gene expression in the whole animal. By the early 1980s, it was possible to alter expression of specific genes in mammals by microinjecting DNA into eggs to create transgenic mice. Rubin and Spradling, who were working on the fruitfly Drosophila melanogaster, decided to take a Trojan-horse approach to the same problem. They took a P element — a natural transposon vector that is known to jump in and out of the fly genome at will — and asked whether it could be used as a vehicle to deliver genes of interest into the genome. In 1982, they showed that this approach did indeed work: a P element containing the rosy eye colour gene could permanently restore normal eye colour in embryos lacking this gene. The main advantage over previous microinjection techniques was that integration of the P element occurred efficiently and was stably inherited by progeny. This basic idea of using P elements as a gateway into the Drosophila genome formed the foundation of most gene manipulation techniques that have since been developed in the fly: this includes the development in the 1990s of the FLP-FRT system of mosaic clone analysis by Golic, and valid user ID Xu and Rubin, and the Gal4 system by Brand and Perrimon — two key techniques for disrupting gene expression in a small subset of cells, and for expressing genes ectopically in your cells of choice.

Meanwhile, in the mouse, Capecchi and It was the single Smithies were both busy trying to tackle the discovery that … made challenge of engineering specific mutations Drosophila an organism into any gene. The approach they took was that could take advantage of advances in to take advantage of the homologous recombinant DNA recombination machinery in cells and to make technology and apply vectors to target a gene of interest. In 1986, them to classical genetics Smithies rescued a mutation in the human and embryology beta-globin locus, and in 1987, Capecchi succeeded in showing that the Hprt recalls Matthew Freeman (hypoxanthine phosphoribosyl transferase) gene could be knocked out in embryonic stem (ES) cells. This revolutionary technique had the potential to be used to introduce any type of mutation in every gene and for the mutant cells to then be stably propagated. Mutant ES cells could, in turn, be used to generate germ-line chimaeras, thereby allowing targeted knockouts or insertions, which could alter the expression of particular genes in the whole animal. 133/http://ad.doubleclick.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730712029?

One challenge in making mouse knockouts even today is the time they can take to produce. It was a method initially observed in plants and then developed in the worm — RNA interference — that now provides the most rapid means to inhibit gene expression in mammals. In 1998, Fire and Mello were investigating the potency of different antisense RNAs to suppress gene expression, and inadvertently discovered that double- stranded RNAs were far more effective. Reminscent of dsRNA viruses that suppress gene expression in plants, this technique required only a few molecules of dsRNA and was inherited non-stoichastically. At the time, they speculated that the underlying mechanism might entail a catalytic amplification process. Over the past 6 years, we have learnt that RNA interference works by hijacking a molecular pathway present from flies to mammals. Its success has now spread to mammals, following the realization that chopping the RNAs into smaller fragments (small interfering RNAs or siRNAs) largely prevents an unwanted interferon response. Although it remains imperative that care be taken to demonstrate specificity in siRNA experiments, it is proving to be the most rapid and universal technique for inhibiting gene expression. In addition, it is now becoming clear that another regulatory mechanism that also involves small RNAs — the microRNA pathway — affords an additional level of control over gene expression used by the organism itself, although the breadth of its functions during development is only now being realized.

REFERENCES

ORIGINAL RESEARCH PAPERS Rubin, G. M. & Spradling, A. C. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353 (1982) PubMed Thomas, K. R. & Capecchi, M. R. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 6, 503–512 (1987) Article Smithies, O. et al. Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317, 230–234 (1985) PubMed Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998). Article PubMed

FURTHER READING Brinster, R. L. et al. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27, 223–231 (1981) Article PubMed Mansour, S. L. et al. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348– 352 (1988) Article PubMed Golic, K. G. Site-specific recombination between homologous chromosomes in Drosophila. Science 252, 958–961 (1991) PubMed Xu, T. & Rubin, G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237 (1993) PubMed Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993) PubMed Gilbert, S. F. Developmental Biology 7th edn: 100–104; 118–120 (2004) FREE

Milestone 13 (1980)

1 July 2004 | doi:10.1038/nrn1461

How the fruit fly gets its stripes Rebecca Barr, Supervising Copy Editor, Nature Research Journals In the fruit fly Drosophila melanogaster, mutations that produced odd- looking or inviable flies were known early in the twentieth century, but it was several decades before their nature began to be understood. Working at the European Molecular Biology Laboratory (Heidelberg, Germany) in the late 1970s, Christiane Nüsslein-Volhard and Eric Wieschaus were among the first to use such mutations to study a complex dilemma: how a multicellular animal develops from a single fertilized egg. At that time, the idea that mutations confer discrete, generally reproducible phenotypes was less established than it is now, and many biologists doubted it was possible to separate out the actions of individual genes during development. Nonetheless, Nüsslein-Volhard and Wieschaus set out systematically to identify mutations relating to one aspect of fly development: the division of the embryo into a linear series of segments. In work that later garnered them the 1995 Nobel prize in Physiology and Medicine (along with Edward Lewis, an earlier pioneer of developmental genetics), Nüsslein-Volhard and Wieschaus treated flies with mutagens to generate a large number of offspring carrying independent mutations — affecting most of the genes in the fly genome. They mated the flies to produce descendants with two copies of each mutation and looked at larvae under a microscope to identify malformations that were caused by aberrant segmentation. At the blastoderm stage, fly embryos become organized into 14 segments. By the time larvae hatch, each segment has a band of backward-pointing projections, called denticles, at its front end, whereas the back end is naked. Nüsslein-Volhard and Wieschaus used these features to distinguish individual segments and identify abnormal patterns of segmentation. By recombination mapping and complementation testing, they mapped the underlying mutations to 15 genes and found that they fitted into three distinct categories: segment-polarity, pair-rule and gap mutations. Segment-polarity mutations cause deletion of parallel parts of each segment. Pair-rule mutations (for instance, even skipped) cause deletions in alternate segments. Gap mutations cause deletion of groups of adjacent segments, corresponding to structurally distinct areas (for instance, the abdomen). The mutant embryos were identifiable shortly after the start of gastrulation, indicating that segmentation genes are active early in development. What did these patterns of mutations say about the sequence of developmental events? Nüsslein-Volhard and Wieschaus proposed that gap genes help establish large-scale body patterns, whereas the segment-polarity and pair-rule genes control segmentation. The two- segment expression pattern of pair-rule genes, they suggested, could reflect an initial segmentation into seven double segments that later divide in half — perhaps avoiding errors that could arise in dividing the relatively few cells of the blastoderm evenly into 14 segments.

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730716078? Later research showed that the fruit fly body plan is dictated by both maternal proteins and differential responses of embryonic genes to those proteins. The maternal proteins establish patterns of polarity in the unfertilized egg that carry forward into the embryo. Interactions between maternal and embryonic genes control location-specific cell differentiation to generate the larval and adult body plans. Researchers have analysed the relationships between segmentation proteins to reveal complex regulatory interactions, generally bearing out Nüsslein-Volhard and Wieschaus's initial theories. Gap proteins are expressed within 2–3 hours after fertilization and divide the embryo into large areas along the head–tail axis. They also serve as transcription factors to regulate the expression of pair-rule genes. Pair-rule proteins set up boundaries within these areas, creating seven fundamental regions, and they also regulate other pair-rule genes or segment-polarity genes. Finally, segment-polarity proteins, which include transcription factors and cell signalling proteins, divide each of the seven regions into two to form the 14 larval segments. The role of segmentation genes is not confined to early embryogenesis — pair-rule genes, in particular, show complex patterns of expression later in development, and they help to control the differentiation of numerous body parts and tissues, the list of which continues to grow.

REFERENCES ORIGINAL RESEARCH PAPER Nüsslein-Volhard, C. & Wieschaus, E. Mutations affecting segment number and polarity in Drosophila. Nature 287, 795–301 (1980) PubMed

FURTHER READING Nüsslein-Volhard, C. et al. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. I. Zygotic loci on the second chromosome. Roux's Arch. Dev. Biol. 193, 267–282 (1984) Jürgens, G. et al. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. II. Zygotic loci on the third chromosome. Roux's Arch. Dev. Biol. 193, 293–295 (1984) Nüsslein-Volhard, C. et al. Mutations affecting the pattern of the larval cuticle in Drosophila melanogaster. III. Zygotic loci on the X-chromosome and fourth chromosome. Roux's Arch. Dev. Biol. 193, 296–307 (1984) Brody, T. The Interactive Fly: gene networks, development and the Internet. Trends Genet. 15, 333–334 (1999) Article PubMed Held, L. I. Jr. Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation (Cambridge Univ. Press, Cambridge, UK, 2002) Gilbert, S. F. Developmental Biology 7th edn: 283–285 (2004) FREE

WEB SITES The Interactive Fly: http://sdb.bio.purdue.edu/fly/aimain/1aahome.htm

1 July 2004 | doi:10.1038/nrn1462 A direct link Rachel Smallridge, Senior Editor, Nature Reviews Molecular Cell Biology Embryonic stem (ES) cells — undifferentiated cells in early embryos that have the ability to form practically any cell type — are present transiently during embryogenesis. Because of their broad developmental potential, researchers knew that such cells could provide a wealth of information about early mammalian development. The problem was how to establish progressively growing cultures of these cells in vitro. Until the 1980s, attempts to obtain such cultures directly from embryos were unsuccessful, and researchers instead studied related cells that were obtained from teratocarcinomas. These tumours arise when normal 1–7.5-day-old mouse embryos are transplanted to an extra- uterine site. Work with teratocarcinoma stem-cell lines provided information on the culture conditions that are suitable for growing pluripotent cells derived from embryos, albeit indirectly. However, a breakthrough came in 1981, when two independent labs reported the establishment of pluripotent ES cell lines directly from mouse embryos. Cells from late pre-implantation-stage embryos (blastocysts) seemed likely to give rise to pluripotent cells in culture, but such embryos contain only a small number of cells. In Nature, Martin Evans and Matt Kaufman showed that, by delaying implantation, they could obtain slightly enlarged mouse blastocysts, and that cells from these blastocysts could be used to establish ES cell cultures. These cultured cells were capable of differentiating in vitro and in vivo. Reporting in Proceedings of the National Academy of Sciences, Gail Martin used a different approach that avoided in vivo alteration. She reasoned that an ES cell line might be obtained by culturing cells isolated from blastocysts in a medium that had previously been conditioned by an established teratocarcinoma stem-cell line (such a medium might contain a factor that stimulates ES-cell proliferation and/or suppresses their differentiation). Using this approach, she established a cell line directly from normal pre-implantation mouse embryos and confirmed its pluripotency by showing that individual cells of this line could differentiate to form a wide variety of cell types in vitro and in vivo. These papers made it possible to obtain pluripotent ES cells directly from embyros, including strains that carried interesting mutations. They also provided a method to generate “...new, genetically marked pluripotent cell lines that can be used for studying various aspects of early mammalian development”. A further breakthrough came in 1998, when, in Science, James Thomson et al. reported the derivation of ES cell lines directly from human blastocysts. After 4–5 months of undifferentiated proliferation in vitro, these cells could still differentiate to form various cell types. Such cell lines will be invaluable to the study of human developmental biology and — once this development is more fully understood — for the generation of specific cell k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730720100? types for transplantation and drug-discovery research. As these authors commented, “Progress in basic developmental biology is now extremely rapid; human ES cells will link this progress even more closely to the prevention and treatment of human disease”. REFERENCES

ORIGINAL RESEARCH PAPERS Evans, M. J. & Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981) PubMed Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634–7638 (1981) PubMed Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998) Article PubMed

FURTHER READING Donovan, P. J. & Gearhart, J. The end of the beginning for pluripotent stem cells. Nature 414, 92–97 (2001) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 100–101; 708–711 (2004) FREE

1 July 2004 | doi:10.1038/nrn1463 131 corpses and a Nobel prize Marie-Thérèse Heemels, Senior Editor, Nature Pruning of cells helps to shape organs, remove a tadpole's tail and carve out fingers or toes. In essence, cell death is necessary to develop gracefully — even for worms. With only 959 cells, a glass-like appearance and a wriggling body, it might seem short of sophisticated features, but over the past two decades the nematode Caenorhabditis elegans has wormed its way into the textbooks by showing how to kill a cell and dispose of the body efficiently. Scientists had previously noticed dying cells that looked different from necrotic cells in various tissues. On the basis of shared morphological features among these deaths, Alastair Currie, John Kerr As seen in Hilary Ellis' and Andrew Wyllie proposed the existence original genetic screen. Top of a conserved, endogenous cell-death panel, C.elegans with active programme, which they dubbed ced-3; bottom panel, 'apoptosis'. But it wasn't until the mid- C.elegans with mutant ced-3. 1980s that Robert Horvitz and colleagues Programmed cell deaths opened their cans of worms to test this occur only with active ced-3 idea comprehensively. By that time, John (arrows in top panel). Arrowheads indicate marker Sulston had mapped out cell lineages in C. cells in both strains. elegans (see Milestone 10), and he Image courtesy of H. R. observed that in every worm, out of 1,090 Horvitz. newborn cells, the same 131 cells die during development, resulting in a nematode of 959 cells exactly. To identify genes that control the fate of the 131 doomed cells, Horvitz and colleagues screened for worms that — after mutagenesis of their genome — contained 'un-dead' cells (that is, cells that should have died, but survived instead). Tracing the mutations in these worms led them to two genes, ced-3 and ced-4 (called ced for cell death abnormal), which were both essential for cell death, and one, ced-9, which prevented death in cells that needed to survive. These experiments established a genetic basis for programmed cell death. Moreover, they showed that most developmental deaths are cell-autonomous — thereby establishing suicide rather than murder as the cause of death. The ced-4 gene was found to encode a protein without any known direct relative, but the cloning of ced-3 provided some immediate insights into the cell's suicide gear. The CED-3 protein was a homologue of a newly discovered mammalian protease, indicating that it caused programmed cell death by clipping other proteins. Along with others, Horvitz and colleagues reported that ced-9, which blocks cell suicide, was a functional homologue of Bcl-2 — a gene that was found to be deregulated in follicular lymphoma cells. By now, Horvitz and colleagues were persuaded that programmed cell death, and its molecular machinery, was conserved between nematodes and mammals, and therefore of very ancient origin. After this, the research on apoptosis snowballed markedly. It soon became clear that in most, if not all, worm cells, the CED-9 protein deters CED-4 from activating CED-3, thereby avoiding accidental deaths. CED-3 k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730724397? was just the first of a series of proteases — collectively called caspases — that were found to be indispensable for efficient apoptosis of animal cells. Also, CED-9 turned out to have many siblings, some of which prevent, whereas others promote, programmed cell death. Many more proteins since have owned up to a role in apoptosis. In a relatively short time we have moved from worm to clinic, as too little or too much apoptosis can result in pathology — not only developmental defects, but also neurodegeneration, autoimmune disease or cancer. Scientific progress frequently happens by serendipity, but here a visionary must have been at play. REFERENCES

ORIGINAL RESEARCH PAPERS Ellis, H. M. & Horvitz, H. R. Genetic control of programmed cell death in the nematode C. elegans. Cell 44, 817–829 (1986) Article PubMed Hengartner, M. O. et al. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356, 494–499 (1992) Article PubMed Yuan, J. et al. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme. Cell 75, 641–652 (1993) Article PubMed Hengartner, M. O. & Horvitz, H. R. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76, 665–676 (1994) Article PubMed

FURTHER READING Gilbert, S. F. Developmental Biology 7th edn: 164–166; 538–539 (2004) FREE

Milestone 16 (1986)

1 July 2004 | doi:10.1038/nrn1464 Morphing heads Sarah Greaves, Assistant Publisher, Nature One classic theme in developmental biology is the idea of a morphogen. Morphogens have long been proposed to act autonomously, defining cell fates at different concentration levels, thereby generating positional information from a gradient (see Bicoid protein is expressed at Milestone 4). However, it wasn't until the anterior of the embryo. pioneering work by Fronhofer and Image courtesy of Valérie Nüsslein-Volhard in 1986, and Driever Schaeffer, New York University, USA. and Nüsslein-Volhard in 1988, that the first classic morphogen in Drosophila melanogaster, Bicoid (Bcd), was identified. Bcd is a maternal effect gene involved in anterior development in the fruitfly, and it controls the expression of zygotic segmentation genes, such as hunchback, in the developing embryo (see Milestone 13). Embryos from Bcd−/− mothers develop without heads or other anterior structures, and Fronhofer and Nüsslein-Volhard demonstrated the clear rescue of this phenotype by transplanting wild-type anterior cytoplasm into mutant embryos. Results of this classic developmental biology experiment indicated that anterior cytoplasm was sufficient to induce anterior development and suppress posterior development wherever it was injected into the mutant embryos. The fact that Bcd mRNA could act over a long range to induce anterior development and suppress posterior development was later confirmed by Driever and Nüsslein-Volhard. Using rabbit polyclonal antibodies to Bcd, Driever and Nüsslein-Volhard identified, in 1988, a 55 kDa protein that is localized in a visible gradient (with the highest concentration at the anterior) within the nuclei of cleaving embryos. This seminal paper was the first to identify a protein gradient in Drosophila embryos and led the authors to conclude that the protein was indeed a morphogen which had long-range effects on neighbouring cells. An accompanying manuscript from the same authors provided back-up genetic evidence, which demonstrated, correlatively, that Bcd was indeed a classic morphogen.

Subsequent work has indicated that Bcd, I believe this was the which was known to contain a homeodomain first really solid molecular and control zygotic gene expression, binds evidence for the much- directly to DNA. Using P-element germ-line discussed but controversial ideas about transposition, Driever, Thoma and Nüsslein- morphogen gradients. Volhard showed how Bcd protein could generate a gradient — the target gene Matthew Freeman hunchback has both high- and low-affinity binding sites for Bcd, which ensure gene expression even at low levels of Bcd and hence generate a gradient. This small collection of manuscripts indicates what underlies a simple idea of a morphogen in vivo to ensure that a fly embryo knows its head from its tail. Work on morphogens wasn't limited to Drosophila, and more recent and controverisal work in Xenopus has indicated that the TGF-β/activin-A family member XTC-MIF, also acts as a classic morphogen in mesoderm development, leading to instructive responses at different protein levels. All of these papers demonstrate how classic ideas were confirmed by a k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730728359? combination of traditional developmental biology transplantation experiments, genetic manipulation and molecular biology, allowing us to appreciate the beauty of a simple idea in action. Even now, 15 years after their publication, these papers form a cornerstone of developmental biology teaching and bring theories on morphogens to life. REFERENCES

ORIGINAL RESEARCH PAPERS Frohnhofer, H.G. & Nüsslein-Volhard, C. Organisation of anterior pattern in the Drosophila embryo by the maternal gene bicoid. Nature 324, 120–125 (1986) Driever, W. & Nüsslein-Volhard, C. A gradient of bicoid protein in Drosophila embryos. Cell 54, 83–93 (1988) Article PubMed Driever, W. & Nüsslein-Volhard, C. The bicoid protein determines position in the Drosophila embryo in a concentration-dependent manner. Cell 54, 95–104 (1988) Article PubMed Driever, W. et al. Determination of spaital domains of zygotic gene expression in the Drosophila embryo by the affinity of binding sites for the bicoid morphogen. Nature 340, 363–367 (1989) Article PubMed

FURTHER READING Green, J. B. A. & Smith, J. C. Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature 347, 391–394 (1990) Article PubMed Chen, Y. & Schier, A. F. The zebrafish Nodal signal Squint functions as a morphogen. Nature 411, 607–610 (2001) Article PubMed Gurdon, J. B. & Bourillot, P. -Y. Morphogen gradient interpretation. Nature 413, 797–803 (2001) Article PubMed Lawrence, P. A. Morphogens: how big is the big picture? Nature Cell Biol. 3, E151–E154 (2001) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 63–68; 272–276 (2004) FREE

WEB SITES Christiane Nüsslein-Volhard’s laboratory: http://www.eb.tuebingen.mpg.de/dept3/home.html

Milestone 17 (1987)

1 July 2004 | doi:10.1038/nrn1465

Two faces of the same coin Sowmya Swaminathan, Associate Editor, Nature Cell Biology Today, it is unremarkable that uncontrolled cell proliferation or cancer is the outcome of a developmental process gone awry. Just over 15 years ago, however, the initial finding that regulators of cell growth were also important for normal development, and that these two processes were driven by common signalling pathways, created a stir both within and outside the developmental biology community. In 1987, a series of papers reporting the sequences of developmentally important genes, such as sevenless and decapentaplegic (Dpp), provided some of the earliest molecular links between development and cancer. Genetic studies in Drosophila melanogaster had already shown that the sevenless gene was required for proper differentiation of photoreceptors in the developing eye. At this time, it was generally appreciated that cell–cell interactions were fundamentally important for developmental processes, and the product of the sevenless gene was predicted to translate information from cell–cell contacts into the appropriate developmental outcome. But the molecular nuts and bolts of how this was achieved remained obscure. After sequencing the sevenless gene, Gerry Rubin and colleagues discovered that it encoded a predicted transmembrane protein bearing a tyrosine kinase domain that is highly related to domains that are present in viral oncogenes and hormone receptors. Based on this homology, they correctly prophesized that the molecular mechanisms of signalling would be the same in both cases — a prediction that came to fruition a few years later. As other cloning and sequencing studies were published in the same year, the connections between development and growth control became clearer. Dpp, which specifies dorso ventral patterning in the fruit fly, and Vg1, a maternal transcript in frog eggs, were both found to encode members of the transforming growth factor-β (TGF-β) family of growth regulators. In another groundbreaking paper from the Nusse laboratory, published in the same year, the cancer–development link was extended to mammals. These authors set out to clone the Drosophila melanogaster homologue of the mouse mammary oncogene, int-1, by genomic hybridization, and they showed that it was identical to the Drosophila patterning gene wingless. They speculated that the int-1 oncogene might shift the balance between proliferation and differentiation in mouse epithelial cells and changes in this homeostasis might lead to tumorigenesis. Although these early studies did not reveal the molecular basis for these observations, what emerged quite clearly was that the overlap between cancer, growth-factor signalling and developmental regulation was a fundamental characteristic of cell division and differentiation.

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730732247? Building on their earlier work on sevenless, Rubin and colleagues carried out a genetic screen to identify mutations that enhanced the phenotype associated with sevenless mutations, hoping that this approach would allow them to identify components of its signalling pathways. In 1991, Michael Simon, along with colleagues from the Rubin laboratory, reported that two of the isolated enhancers of sevenless encoded known signalling molecules — the GTPase Ras, and its guanine nucleotide exchange factor, Cdc25. Ras was not only a characterized oncogene that was known to have transforming ability, but it had also been shown to function downstream of other protein tyrosine kinases and to regulate development in the worm. As predicted 4 years earlier, the parallels between sevenless and protein tyrosine kinases seemed to extend to their signalling pathways. At this stage, however, the possibility remained that Ras might influence sevenless through an independent pathway, and conclusive proof that Ras acts in the sevenless pathway only came with subsequent biochemical analysis. However, biochemical links between Ras and other receptor tyrosine kinases did exist, so these genetic data also supported a physiological role for Ras in receptor tyrosine kinase signalling. During the past decade, the number of developmental regulators that are also associated with cancer has multiplied, as has our understanding of how these processes are intertwined. These early studies — a testament to the combined predictive power of genetics and sequence homology — were profoundly influential in establishing a conceptual framework for considering cancer and development as related processes or as 'two faces of the same coin'.

REFERENCES

ORIGINAL RESEARCH PAPERS Hafen, E. et al. Sevenless, a cell-specific homeotic gene of Drosophila encodes a putative transmembrane receptor with a tyrosine kinase domain. Science 236, 55–63 (1987) PubMed Rijsewijk, F. et al. The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell 50, 649–657 (1987) Article PubMed Simon, M. A. et al. Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signalling by the sevenless protein tyrosine kinase. Cell 67, 701–716 (1991) Article PubMed

FURTHER READING Padgett, R. W. et al. A transcript from a Drosophila pattern gene predicts a protein homologous to the transforming growth factor-beta family. Nature 325, 81–84 (1987) Article PubMed Weeks, D. L & Melton, D. A. A maternal mRNA localised to the vegetal hemisphere in Xenopus eggs codes for a growth factor related to TGF-β. Cell 51, 861–867 (1987) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 621–622; 703–705 (2004) FREE

1 July 2004 | doi:10.1038/nrn1466

An unequal divide Deepa Nath, Senior Editor, Nature A fundamental dilemma in development is unravelling how a single cell can divide asymmetrically to generate two daughter cells with distinct fates. Thanks to pioneering studies that began in the 1980s, we now know that an important mechanism for generating differences between daughter cells is the unequal distribution of developmentally important molecules or determinants. Our knowledge of the machinery that directs asymmetric cell division has come largely from experiments using the nematode worm Caenorhabditis elegans and the fruitfly Drosophila melanogaster. The worm has proved to be particularly useful in this respect, because its early development is characterized by a cascade of asymmetric divisions. Indeed, the very first division of the single-cell C. elegans embryo is asymmetric — the cell (P0) divides along its anterior–posterior axis to produce a larger AB cell and a smaller posterior P1 cell.

Before cell division, an axis of A landmark paper, published in 1988 by polarity is established, which Kemphues et al., identified genes that causes differing concentrations are involved in cell-fate specification in C. of determinants that induce elegans. A study of maternal-effect lethal different cell fates in the daughter cells. mutations led to the identification of the par (partitioning defective) genes par1–4. Wild-type C. elegans oocytes contain a uniform distribution of P granules — large ribonuclear particles that normally segregate into P1. Mutations in the four par genes led to abnormal positioning of the mitotic spindle and aberrant localization of the P granules, leading the authors to propose that the par genes encode products that are required for spindle placement and cytoplasmic localization. In the mid 1990s, Kemphues and colleagues cloned the C. elegans par-1 and par-3 genes. PAR-1 is a conserved serine/threonine kinase, whereas PAR-3 turned out to be a novel protein. Both of these proteins are asymmetrically distributed in the zygote: PAR-1 is enriched at the posterior periphery, whereas PAR-3 is found at the anterior periphery. In early germ-line precursor cells, PAR-1 localization correlates strikingly with P granule distribution and requires the kinase activity of PAR-1. Meanwhile, the distribution of PAR-3 controls spindle orientation. PAR-3 localization depends on the par-2 gene, and PAR-3 is required for PAR-1 localization.

re.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730736213? Experiments in Drosophila uncovered additional asymmetrically segregating cell-fate determinants. In the developing peripheral nervous system, sensory organ precursors (SOPs) divide symmetrically to give the secondary precursors IIa and IIb. These precursors then divide asymmetrically — IIa produces cells that form the hair and socket, whereas IIb generates cells that differentiate into the neuron and sheath. A breakthrough in elucidating the underlying molecular mechanism came from the Jan laboratory. In a 1994 paper, they identified a protein in Drosophila sensory organs called Numb, which is localized within the SOP before cell division and segregates to only one of the daughter cells during division. So, is Numb necessary to give IIa and IIb distinct fates? Overexpression of Numb during SOP division results in the formation of two IIb cells rather than the normal IIa/IIb pair. Furthermore, in the absence of Numb, IIb cells differentiate inappropriately, forming cells that normally derive from IIa. These results demonstrated that the asymmetric segregation of a determinant during cell division could induce a specific fate in one of the two daughter cells. In the developing fly central nervous system, neuroblasts divide asymmetrically to produce an apical daughter that remains a neuroblast and a smaller basal daughter called a ganglion mother cell. Knoblich and colleagues showed that expression of the inscuteable gene is required for asymmetric segregation of Numb, as well as correct spindle orientation, in fly neuroblasts and epithelial cells. The Inscuteable protein localizes to the apical-cell cortex before mitosis and precedes the basal localization of Numb. These results indicated that asymmetric localization of Inscuteable establishes positional information for both spindle orientation and asymmetric localization of Numb. The Par proteins have also been shown to be involved in asymmetric Numb localization in neuroblasts. Together, these pioneering papers demonstrated that asymmetric distribution of cellular determinants underlies the generation of cellular diversity, and researchers are now trying to establish whether similar principles determine asymmetric cell division in vertebrates.

REFERENCES ORIGINAL RESEARCH PAPERS Kemphues, K. J. et al. Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52, 311–320 (1988) PubMed Guo, S. & Kemphues, K. J. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611– 620 (1995) Article PubMed Etemad-Moghadam, B. et al. Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 83, 743–752 (1995) Article PubMed Rhyu, M. S. et al. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells. Cell 76, 477–491 (1994) Article PubMed Kraut, R. et al. Role of inscuteable in orienting asymmetric cell divisions in Drosophila. Nature 383, 50–55 (1996) Article PubMed

FURTHER READING Knoblich, J. A. Asymmetric cell division during animal development. Nature Rev. Mol. Cell Biol. 2, 11–20 (2001) Article PubMed Jan, Y.-N. & Jan, L. Y. Asymmetric cell division in the Drosophila nervous system. Nature Rev. Neurosci. 2, 772–779 (2001) Article PubMed

1 July 2004 | doi:10.1038/nrn1467

Chasing the elusive inducer Annette Markus, Associate Editor, Nature Neuroscience For more than six decades, scientists around the world searched for the molecular basis of neural-plate induction, without much luck. Back in 1924, Spemann and Mangold had shown that a speck of tissue from the dorsal lip of the early newt gastrula could induce a second neural plate when transplanted into an ectopic site. This group of dorsal lip cells, the 'organizer', developed into mesodermal notochord and induced neural fates in the overlying ectoderm (see Milestone 1). The standard hypothesis, arising from this and subsequent experiments, postulated that the organizer and resulting notochord, through secreted soluble molecules, instruct neural-plate differentiation in the overlying ectoderm. This straightforward idea The Xenopus experiments that demonstrated the default model took a few knocks over the years as it of neural induction became apparent that a bewildering array of substances, and even physical manipulation of early embryos, could induce ectopic neural differentiation, and none of these were even remotely plausible candidates for an organizer signal. Nevertheless, this hypothesis reigned into the 1980s. Then, in 1989, Grunz and Tacke published a surprising observation. It was known that isolated early ectoderm explants differentiated into epidermal tissue in vitro, but what if normal cell–cell interactions were disrupted? If the cells were dissociated then randomly reaggregated immediately, the result was again epidermis. But if reaggregation was delayed for an hour or more, neural markers began to be expressed, and if dissociated ectodermal cells were kept in suspension for 5 hours before reaggregation, they differentiated into neural tissue exclusively. This indicated that the absence, not the presence, of an intercellular signal was necessary for neural differentiation. Neural fate might indeed be the 'default' fate of ectodermal cells. It was time for molecular biologists to step into the fray. Smith and Harland developed an assay to identify mRNAs that coded for proteins that could induce a neural plate in ventralized Xenopus embryos. They generated and systematically screened a library of mRNAs derived from hyperdorsalized gastrula-stage embryos. In 1992, they isolated noggin, a novel mRNA that could not only induce a neural plate (and complete dorso–ventral axis) without requiring the presence of mesoderm, but also was expressed appropriately in the dorsal lip and in the notochord. However, the mechanism of how noggin caused neural-plate formation, and how it fitted into the new 'default hypothesis', remained obscure. The default hypothesis was bolstered by Hemmati-Brivanlou and Melton, who reported in 1994 that inhibition of the activin II receptor could, by itself, induce neural differentiation in the absence of the notochord. k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730740649? In 1995, at last, the concept of a neural inducer and the idea of neural phenotype as the default outcome of ectodermal differentiation came together. De Robertis and colleagues identified chordin, another new dorsal lip protein that could directly induce a neuraxis. They also showed that bone morphogenetic protein-4 (BMP4), a growth factor related to activin, inhibited the neuralizing activity of both chordin and noggin in Xenopus embryos. The same group soon found that chordin directly binds and inactivates BMP4, and Harland and colleagues reported a similar function for noggin. So, the default hypothesis seemed finally to stand on solid genetic and biochemical ground. It was proposed that neural induction requires the nhibition — by noggin, chordin or other molecules — of the epidermis- nducing, anti-neural BMP4 signal. The search for neural-plate instructive signalling molecules had lasted for seven decades and ended with the recognition that the instructive signal as such did not exist. This is not the end of the story, however. Recent findings indicate that the neural ectoderm is specified in the blastula, before the Spemann organizer even forms. Fibroblast growth factor (FGF) signalling is required at this stage to enable later neural differentiation. In Xenopus at least, the anterior — but not the posterior — neural ectoderm is specified at the blastula stage, through a mechanism that already involves noggin and chordin. These and other studies are now elucidating the genesis of the default' state.

REFERENCES

ORIGINAL RESEARCH PAPERS Grunz, H. & Tacke, L. Neural differentiation of Xenopus laevis ectoderm takes place after disaggregation and delayed reaggregation without inducer. Cell Diff. Dev. 28, 211–218 (1989) Article Smith, W.C. & Harland, R. M. Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell 70, 829–840 (1992) Article PubMed Hemmati-Brivanlou, A. & Melton, D. A. Inhibition of activin receptor signaling promotes neuralization in Xenopus. Cell 77, 273–281 (1994) Article PubMed Sasai, Y. et al. Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature 376, 333–336 (1996) Article Piccolo, S. et al. Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589–598 (1996) Article PubMed Zimmermann, L. B. et al. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein-4. Cell 86, 599–606 (1996) Article PubMed Streit, A. et al. Initiation of neural induction by FGF signalling before gastrulation. Nature 406, 74–78 (2000) Article PubMed Kuroda, H. et al. Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin and Cerberus. PLoS Biol. 2, 624–634 (2004)

FURTHER READING Muñoz-Sanjuán, I. & Hemmati-Brivanlou, A. Neural induction, the default model and embryonic stem cells. Nature Rev. Neurosci. 3, 271–280 (2002) Article PubMed De Robertis, E. M. & Sasai, Y. A common plan for dorsoventral patterning in Bilateria. Nature 380, 37–40 (1996) PubMed Gilbert, S. F. Developmental Biology 7th edn: 327–337; 362–363 (2004) FREE

Milestone 20 (1990)

1 July 2004 | doi:10.1038/nrn1468 The keys to sex Myles Axton, Editor, Nature Genetics Of the genes that are responsible for decisions in mammalian development, two on the sex chromosomes stand out as key molecular switches. The Y-linked Sry gene encodes a DNA-binding protein that specifies male gender. The X-linked Xist encodes an untranslated RNA that controls X-inactivation. Genetic analysis of Sry and Xist has revealed phenotypes of mutational loss-of-function and transgenic gain-of-function that established these two genes as both necessary and sufficient for their respective roles. An idea about the location of the gene that specifies testes, and subsequent male development, was decisive to its isolation. It had to lie in the region where the sequence of the Y chromosome is unique, but close enough to the region shared with the X chromosome to account for the accidental, albeit rare, crossover with the X chromosome to generate XX males or XY females. Peter Goodfellow, Robin Lovell-Badge and colleagues showed that SRY, which is localized within 35 kb of the boundary of the Y-unique sequence on the human Y chromosome, encodes a testis-specific protein that is similar to other DNA-binding HMG- box-containing proteins. Around the same time, Philippe Berta, Goodfellow, Marc Fellous and colleagues, and Gerd Scherer and colleagues, found sex-reversed XY female humans who carried mutations in the SRY gene, thereby demonstrating that it was necessary for maleness. The Lovell-Badge and Goodfellow teams then proved Sry to be sufficient for testis development by showing that an Sry transgene conferred a male phenotype on XX mice. The transgenic mice were infertile, however, as the XX genotype is incompatible with sperm production. This result indicates that the sex chromosomes are important for both sex determination and gamete production, but that the two functions are independently regulated. Tortoiseshell cats are a common reminder that most female mammals are mosaics containing clusters of cells that express the X chromosome inherited from their father, whereas others are expressing the mother's X chromosome. This phenomenon — termed dosage compensation by X- inactivation — was first described by Mary Lyon in the mouse. It ensures that most genes on the X chromosome are expressed at similar levels in males and females. The first clues to what underlies this process came from Huntington Willard and colleagues, who discovered that the XIST gene is only expressed from the inactive human X chromosome, so it presumably acts in cis on the chromosome that produces the XIST RNA. Neil Brockdorff, Sohaila Rastan and colleagues found the same for Xist in mouse, and went on to identify the 15 kb untranslated nuclear transcript that is conserved in sequence and structure between mice and humans. The targeted knockout of Xist in XX mouse embryonic stem (ES) cells showed conclusively that the gene is essential for X-inactivation. In eutherian mammals, X-inactivation is random, but in marsupials, imprinting ensures that the paternal X is preferentially inactivated. The same also happens in extra-embryonic tissues of eutherian mammals, and probably in pre-implantation mouse embryos too. A chromosome-counting mechanism seems to allow inactivation of all but one X chromosome k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730744904? during development. By studying transgenic ES cells and embryos harbouring deletions of sequences in the antisense strand of the DNA, Jeannie Lee showed that Xist is regulated by its 40 kb antisense partner, Tsix, which codes for another cis-acting untranslated RNA. It now seems that both the imprinting and counting mechanisms control Xist through Tsix, thereby ensuring that only one X chromosome will remain active in the cells. REFERENCES ORIGINAL RESEARCH PAPERS Sinclair, A. H. et al. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346, 240–244 (1990) Article PubMed Berta, P. et al. Genetic evidence equating SRY and the testis-determining factor. Nature 348, 448–450 (1990) Article PubMed Jäger, R. J. et al. A human XY female with a frame shift mutation in the candidate testis- determining gene SRY. Nature 348, 452–454 (1990) Article PubMed Koopman, P. et al. Male development of chromosomally female mice transgenic for Sry. Nature 351, 117–121 (1991) Article PubMed Lyon, M.F. Gene action in the X chromosome of the mouse (Mus musculus L.) Nature 190, 372–373 (1961) Brown, C.J. et al. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 349, 38–44 (1991) Article PubMed Brockdorff, N. et al. Conservation of position and exclusive expression of mouse Xist from the inactive X chromosome. Nature 351, 329–331 (1991) Article PubMed Brockdorff, N. et al. The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell 71, 515–528 (1992) Article PubMed Kay, G.F. et al. Expression of Xist during mouse development suggests a role in the initiation of X chromosome inactivation. Cell 72, 171–182 (1993) PubMed Penny, G.D. et al. Requirement for Xist in X chromosome inactivation. Nature 379, 131–137 (1996) Article PubMed Lee, J. T. Disruption of imprinted X inactivation by parent-of-origin effects at Tsix. Cell 103, 17–27 (2000) Article PubMed

FURTHER READING Zarkower, D. Establishing sexual dimorphism: conservation amidst diversity? Nature Rev. Genet. 2, 175–185 (2001) Article PubMed Capel, B. Sex in the 90s: SRY and the switch to the male pathway. Ann. Rev. Physiol. 60, 497– 523 (1998) Article Gilbert, S. F. Developmental Biology 7th edn: 551–556; 561–567 (2004) FREE

Milestone 21 (1991)

1 July 2004 | doi:10.1038/nrn1469

Sonic hedgehog, the morphogen Jack Horne, Senior Editor, Nature Cell Biology

A gradient of Shh specifies progenitor domains for motor neurons (MN) and four classes of interneurons (VO–V3)

The process of induction, in which signals from one tissue change the differentiation of another, is a key concept in development. Although observations of inductive processes date from the early embryological literature, it wasn't until the 1990s that the molecular underpinnings began to be defined. A few key papers along the road to understanding how the early central nervous system (CNS) is patterned along the dorsoventral (DV) axis provide an excellent illustration of how the study of development was ushered into the age of molecular biology. Patterning of the CNS along the DV axis — for example, the specification of motor neurons in the ventral half of the spinal cord and sensory neurons in the dorsal half — begins just as the neural plate is folding along its midline to form the neural tube. A series of grafting and ablation experiments by van Straaten and Drukker showed that the notochord — a rod-like structure of mesodermal tissue that is just ventral to the neural tube — could induce the development of a specialized structure along the ventral midline of the neural tube called the floor plate. The floor plate was known to have effects on the guidance of some spinal-cord axons, and, along with the notochord, it was implicated as a signalling centre. Very early on in the 1990s, Jessell and colleagues set out to test whether signals from the notochord or floor plate might be responsible for controlling the DV pattern of neuronal differentiation. Through grafting experiments in the chick embryo, they showed that both the notochord and the floor plate could induce ectopic patterns of ventral motor neuron layers. Furthermore, removal of the notochord or floor plate resulted in the loss of these ventral motor neurons. So, both notochord and floor- plate cells could release signals that are required for the initial DV patterning of the CNS. Induction by notochord and floor-plate explants resulted in distinct subsets of neuron types at defined distances from the ectopic tissue source. The authors proposed a model in which a diffusible signal produced in the notochord or floor plate results in a gradient of the signal across the DV axis, and this gradient is responsible for the pattern of neurons induced. The search began for the molecular identity of the diffusing signal.

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730750157? The next breakthrough came from the cloning of vertebrate homologues of the Drosophila segment polarity gene hedgehog (hh). Two groups simultaneously cloned hh homologues — McMahon and colleagues in the mouse, and Ingham and colleagues in zebrafish. In both cases, the expression pattern of one homologue in particular, sonic hedgehog (Shh), implied that it might be involved in ventral CNS patterning. Shh was expressed in both the notochord and the floor plate at the appropriate times in development that were expected for the inductive signal. Furthermore, ectopic expression of Shh could induce floor-plate-specific genes. Shh was predicted to be a secreted protein, so it was an obvious candidate for the diffusible signal that was predicted by the earlier study of Jessell and colleagues. Subsequent work has shown that differentiation of the floor plate might be more complex, perhaps beginning during gastrulation in the node, but it has also confirmed that Shh is indeed the inductive signal for ventral patterning of the CNS. Furthermore, we now know that a gradient of Shh released by the floor plate is important for this patterning. Distinct neuronal subsets respond to different concentrations of Shh along this DV concentration gradient. Indeed, Shh has come to be known as a classic example of a morphogen — a signal that regulates the spatial pattern of cell differentiation in a concentration-dependent manner — largely due to these groundbreaking experiments on the patterning of the CNS.

REFERENCES ORIGINAL RESEARCH PAPERS Yamada, T. et al. Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord. Cell 64, 635–647 (1991) Article PubMed Echelard, Y. et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75, 1417–1430 (1993) PubMed Krauss, S. et al. A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos. Cell 75, 1431–1444 (1993) PubMed

FURTHER READING van Straaten, H. W. et al. Effect of the notochord on the differentiation of a floor plate area in the neural tube of the chick embryo. Anat. Embryol. 177, 317–324 (1988) PubMed Jessell, T. M. Neuronal specification in the spinal cord: indictive signals and transcriptional codes. Nature Rev. Genet. 1, 20–29 (2000) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 162–163; 401–402 (2004) FREE

1 July 2004 | doi:10.1038/nrn1470 Coordinating development Louisa Flintoft, Assistant Editor, Nature Reviews A key feature of animal development is that the body axes are aligned correctly with respect to each other, indicating that their formation must be tightly coordinated. The first insight into how such coordination is achieved came in 1995 from a paper describing studies in Drosophila carried out by Daniel St Johnston and colleagues. In flies, anterior–posterior (AP) polarity originates when the oocyte moves to the posterior end of the surrounding egg chamber and signals to nearby somatic follicle cells to assume a posterior fate. Later, these follicle cells signal back to the oocyte, causing polarization of the microtubule cytoskeleton. This leads to the localization of bicoid and oskar mRNAs to opposite ends of the oocyte, where these transcripts later direct the development of anterior and posterior features, respectively. St Johnston's group suspected that a gene called gurken might be important in this process. gurken was already known to be involved in establishing dorso–ventral (DV) polarity: localized secretion of gurken protein at the upper side of the oocyte signals to the adjacent follicle cells to assume a dorsal fate, a key step in setting up the DV axis. However, the phenotype of gurken mutants indicated that this gene has an earlier role in determining AP polarity. St Johnston and colleagues confirmed this, showing that gurken is required in the oocyte for the specification of posterior follicle cells. Striking defects in AP polarity were also seen in oocytes in gurken mutants: the normal localization of bicoid and oskar mRNAs to opposite ends of the oocyte was lost. This indicated that gurken signalling sets up the AP polarization of the microtubule cytoskeleton that directs the correct transport of these mRNAs. To test this, the authors used a fusion of kinesin — a protein that normally moves towards the plus ends of microtubules at the posterior end of the oocyte — with β-galactosidase. In gurken mutants, this protein failed to move to the posterior pole, confirming the loss of microtubule polarity. As well as being crucial for setting up the AP axis, microtubule polarization is also essential for DV polarity. The localized secretion of gurken that initiates dorsal follicle-cell fate depends on the movment of the oocyte nucleus along microtubules to the anterior–dorsal corner of the oocyte, leading to the accumulation of gurken mRNA close to the dorsal surface. The fact that this occurs after the posterior follicle cells have been specified indicated that the gurken-dependent microtubule polarization that initiates AP polarity might also be responsible for determining the position of the DV axis. This was confirmed when it was shown that the oocyte nucleus does not migrate to its correct position in gurken mutants. In addition, gurken mRNA fails to localize to the correct position at the anterior–dorsal corner of the oocyte when the gurken receptor in the follicle cells is absent. As St Johnston and colleagues put it in their paper, “It is hard to imagine how two perpendicular axes can be defined unless the orientation of one depends on the other.” This is exactly what this study showed: the body axes in Drosophila are coordinated by the initial establishment of AP polarity, which then determines where the DV axis is formed.

REFERENCES ORIGINAL RESEARCH PAPER Gonzalez-Reyes, A., Elliot, H. & St Johnston, D. Polarization of both major body axes in Drosophila by gurken torpedo signalling. Nature 375, 654–658 (1995) Article PubMed

FURTHER READING Gilbert, S. F. Developmental Biology 7th edn: 263–303 (2004) FREE

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730754528? Milestone 23 (1995)

1 July 2004 | doi:10.1038/nrn1471

A pathway to asymmetry Kyle Vogan, Associate Editor, Nature Genetics Vertebrates appear bilaterally symmetrical. Beneath the surface, however, their internal organs display marked left/right (L/R) asymmetry. To achieve this final body plan, vertebrate embryos must distinguish right from left and transmit this information to individual organs as they develop. How are these early L/R patterning decisions made? A key breakthrough came in 1995 through the pioneering work of Mike Levin, Cliff Tabin and colleagues, who discovered a cascade of genes regulating L/R organ asymmetry in the chick. The starting point for this work was the discovery of a transient asymmetry in Shh expression on the left side of Hensen's node — a midline structure that serves as a key source of patterning signals in the early embryo. Subsequently, Levin et al. stumbled on a second gene, Nodal, that was expressed immediately adjacent to Shh in a domain extending throughout the embryo's left side. Importantly, when Levin et al. misexpressed Shh on the right side of the embryo, Nodal was induced ectopically and the normal rightward bending of the heart tube became randomized. This study revealed, for the first time, the existence of genes with L/R asymmetric expression patterns that could influence the direction of subsequent L/R morphogenetic events. In 1996, follow-up studies led by Liz Robertson and Michael Kuehn showed that this asymmetry in Nodal expression is also present in mouse and Xenopus, identifying Nodal as a central player in a conserved vertebrate L/R pathway. Simultaneously, Hiroshi Hamada and colleagues discovered another gene, which they called Lefty because of its striking expression in the left lateral plate mesoderm and in the left half of the prospective floor plate. The Lefty locus was subsequently found to contain two closely linked and highly related genes, Lefty-1 and Lefty-2. The overlapping expression of Nodal and Lefty-2 in the left lateral plate mesoderm indicated that these genes might act together to impart left- sided identity to developing organs. However, an unexpected role for Lefty genes subsequently emerged from the analysis of Lefty-1 knockout mice: Lefty-1, rather than specifying 'leftness', is actually required for main-taining right-sided identity. As the primary site of Lefty-1 expression is in the left half of the prospective floor plate, Meno et al. proposed that Lefty-1 acts as a midline barrier, preventing left-sided signals, such as Nodal, from crossing the midline and conferring left identity to cells on the right.

k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730759133? Nodal expression is extinguished before the onset of L/R morphogenesis, so how does it influence organ situs? In 1998, studies led by Cliff Tabin, Juan Carlos Izpisúa Belmonte, Sumihare Noji and Marian Ros identified Pitx2 as a key downstream mediator in the L/R pathway. Unlike Nodal, Pitx2 expression is maintained on the left side as organs develop, bridging the gap between asymmetric gene expression and L/R morphogenesis. Still, a key question remained. How is the initial distinction between left and right made? In a landmark 1998 study, Nobutaka Hirokawa and colleagues made the remarkable discovery that the mouse node contains a cluster of motile cilia which generate a leftward flow across the node's ventral surface. As the node is where the earliest L/R asymmetric expression patterns are seen, and directional flow could, in principle, arise from the inherent chirality of the ciliary apparatus without prior input of L/R information, these results indicated that ciliary motion might initiate L/R axis specification. So, in the space of 3 exciting years, we witnessed the birth of a model that could explain, in broad terms, how embryos distinguish left from right and use this information to coordinate the asymmetric shape and positioning of organs along the body's L/R axis.

REFERENCES

ORIGINAL RESEARCH PAPERS Levin, M. et al. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82, 803–814 (1995) PubMed Meno, C. et al. Left-right asymmetric expression of the TGF-family member lefty in mouse embryos. Nature 381, 151–155 (1996) Article PubMed Collignon, J. et al. Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381, 155–158 (1996) Article PubMed Lowe, L. A. et al. Conserved left-right asymmetry of nodal expression and alterations in murine situs inversus. Nature 381, 158–161 (1996) Article PubMed Meno, C. et al. lefty-1 is required for left-right determination as a regulator of lefty-2 and nodal. Cell 94, 287–297 (1998) Article PubMed Yoshioka, H. et al. Pitx2, a bicoid-type homeobox gene, is involved in a Lefty-signaling pathway in determination of left-right asymmetry. Cell 94, 299–305 (1998) Article PubMed Logan, M. et al. The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell 94, 307–317 (1998) Article PubMed Piedra, M. E. et al. Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell 94, 319–324 (1998) Article PubMed Ryan, A. K. et al. Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394, 545–551 (1998) Article PubMed Nonaka, S. et al. Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95, 829–837 (1998) PubMed

FURTHER READING Essner, J. J. et al. Conserved function for embryonic nodal cilia. Nature 418, 37–38 (2002) Article PubMed Nonaka, S. et al. Determination of left-right patterning of the mouse embryo by artificial nodal flow. Nature 418, 96–99 (2002) Article PubMed McGrath, J. et al. Two populations of node monocilia initiate left-right asymmetry in the mouse. Cell 114, 61–73 (2003) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 337; 363–365; 381–383 (2004) FREE

Milestone 24 (1997)

1 July 2004 | doi:10.1038/nrn1472 Time for segmentation Magdalena Skipper, Editor, Nature Reviews Genetics

Hairy1 expression (dark shading) in presomitic mesoderm and newly formed somites in a chick embryo

Most animal body plans are segmented, even if this segmentation is not always externally clear. The first insights into animal segmentation came from Drosophila, surprisingly, although the segmentation cascade identified in the fly did not seem to be conserved in vertebrates, indicating that this process must have arisen independently at least twice. The cloning and analysis of chick Hairy, a homologue of a Drosophila segmentation gene, in 1997 was to challenge this belief. By analyzing Hairy1 expression in the chick presomitic mesoderm (PSM) — a tissue from which the most obviously segemented structure in the vertebrate body (the somites) emerges — Pourquié and colleagues showed that its mRNA appears as a wave that starts at the posterior and moves towards the anterior, and that this wave of expression accompanies formation of each somite. Each cell in the PSM goes through a constant number of Hairy1 oscillations from the time it emerges from the primitive streak to the time when it is incorporated into a somite. This observation provided the first molecular evidence for the existence of a previously postulated molecular clock, or oscillator, that sets the periodicity with which the somite boundaries are laid during vertebrate somitogenesis. Subsequently, it was shown that expression of several Notch pathway components also oscillates in the PSM, establishing a link between Notch signalling and the segmentation clock. More recently, Wnt signalling has also been implicated in the segmentation clock mechanism. The next piece in the puzzle came in 2001 when Pourquié and colleagues provided insights into the mechanisms controlling the spacing of somitic boundaries along the anterior–posterior (AP) axis. Their data indicated that fibroblast growth factor (FGF) signalling translates the pulse of the segmentation clock into the reiterated arrangement of segment boundaries. Pourquié and colleagues showed that Fgf8 forms a dynamic gradient (its concentration being highest in the tail bud) that is translated k.net/adi/milestone.nature.com/development;pos=right;dcopt=ist;lg=no;;sz=120x600;ord=1559730764331? into a graded FGF signalling response, which is used to position the wavefront. Increasing FGF8 concentration in the PSM (for example, by implanting FGF8-soaked beads) increases the number of oscillations that the cells go through in the PSM — cells that go through more oscillations are incorporated into different somites that, on the basis of their Hox gene expression, have a more posterior fate, despite their physical position in the embryo remaining unchanged. This mechanism provides an efficient means to couple the spatio-temporal activation of segmentation to the posterior elongation of the embryo. The link between the segmentation clock and AP patterning was further elaborated by Duboule and colleagues, who showed that Hox genes are expressed in a wave-like pattern in the PSM that depends on Notch signalling. Although many insights into molecular aspects of somitogenesis have come from work on chick, mouse, zebrafish and the frog, the somitogenesis clock is far from well understood. And many questions remain: for example, the nature of the oscillator still remains unknown. Already, in the 1970s, theoretical models were being proposed for how the periodicity of the somites arises — 'clock and wavefront', 'clock and trail', 'cell cycle' and 'Meinhardt's' models being among the more prominent. Most of the exiting data are consistent with the 'clock and wavefront' model, according to which the cells in the PSM oscillate between cellular states in response to an internal oscillator; segmental boundaries form when a maturation wave that progresses caudally passes over cells in a particular phase of the clock. Howerer, the jury is still out on the final decision. Interestingly, recent studies in invertebrates have opened the exciting possibility that the 'clock and wavefront' system characterized in vertebrates might also operate in invertebrates. If true, the system would represent an ancestral segmentation mechanism shared by these two phyla and would bring us full circle in our quest to understand animal segmentation.

REFERENCES ORIGINAL RESEARCH PAPERS Pelmeirim, I. et al. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639–648 (1997) PubMed Dubrulle, J. et al. FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232 (2001) Article PubMed

FURTHER READING Zakany, J. et al. Localized and transient transcription of Hox genes suggests a link between patterning and the segmentation clock. Cell 106, 207–217 (2001) Article PubMed Aulehla, A. et al. Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395–406 (2003) Article PubMed Stollewerk, A. et al. Involvement of Notch and Delta genes in spider segmentation. Nature 423, 863–865 (2003) Article PubMed Saga, Y. & Takeda, H. The making of the somite: molecular events in vertebrate segmenation. Nature Rev. Genet. 2, 835–845 (2001) Article PubMed Gilbert, S. F. Developmental Biology 7th edn: 467–473 (2004) FREE