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Historyandbasicconcepts E The originsof developmentalbiology E A conceptualtool kit The aim of this chapter is to provide a conceptualframework for the study of We startwith a briefhistoryof the studyof embryonicdevelopment, development. biologywerefirst how someof the keyquestionsin developmental whichillustrates Thebig formulated,andcontinuewith someof the essentialprinciplesof'development. questionis how doesa singlecell-the fertilizedegg-give riseto a multicellular into tissuesand organism,in whicha multiplicityof differentcelltypesareorganized body.Thisquestioncanbe studiedfrom many organsto makeup a three-dimensional allof whichhaveto befittedtogethertoobtaina completepicture differentviewpoints, andwhenandwhere;howcellscommuniof dwelopmentwhichgenesareexpressed, fate isdetermined,howcellsproliferate catewith eachother;howa cell'sdevelopmental into specialized celltypes;andhow majorchangesin bodyshapeare anddifferentiate is ultimatelydrivenby the produced.We shallseethat an organism'sdevelopment of its genes,determiningwhichproteinsarepresentin whichcells regulatedexpression Thegenesprovidea andwhen,ln turn, proteinslargelydeterminehow a cellbehaves. generativeprogramfor development,not a blueprint,as their actionsare translated signaling, into developmental outcomesthroughcellularbehaviorsuchasintercellular movement. proliferation, and cell celldifferentiation, cell The development of multicellular organisms from a single cell-the fertilized egg-is a brilliant triumph of evolution. During embryonic development, the egg divides to give rise to many millions of cells, which form structures as complex and varied as eyes, arms, heart, and brain. This amazing achievement raises a multitude of questions. How do the cells arising from division of the fertilized egg become different frorn each othefl How do they become organized into structures such as limbs and brains? What controls the behavior of individual cells so that such highly organized patterns emerge?How are the organizing principles of development embedded within the egg, and in particular within the genetic material, DNA? Much of the excitement in developmental biology today comes from our growing understanding of how genes direct these developmental processes,and genetic control is one of the main themes of this book. Ttrousands of genes are involved in controlling development, but we will focus only on those that have key roles and illustrate general principles. The development of an embryo from the fertilized egg is known as embryogenesis.One of its first tasks is to lay down the overall body plan of the organism, and we shall see that different organisms solve this fundamental Ilr' qillt 2 . 1: HtsroRYANDBAstccoNcEPTs Fig. 1.1 Scanningelectron micrographof the head of an aduft Drosophilomelonogoster. Scalebar= 0.1mm. Photogroph byD. khorfe,fromkiencePhotoLibrory. Fig. 1.2 Photographof a lizard,the south easternfive-linedskink,after it hasreleased its tail in defense.Thisspeciescandeliberately sheditstail asa technique to avoidcaptureby predators;andthen regenerate it. A pieceof discarded tailcanbeseenbelowthe skink. Photogroph Films. fromOxfordScientiftc problem in severalways. The focus of this book is mainly on animal developmentthat of vertebrates such as frogs, birds, fish, and mammals, and of a selection of invertebrates, such as the sea urchin, ascidians, and, above all, the fruit fly Drosoplnlamelarwgaster (Fig. 1.1) and the nematode woran Caenorhabditis elegans.ltis in these last two organisms that our understanding of the genetic control of development is most advanced, and the main features of their early development are considered in Chapters 2 and 5, respectively. In Chapter 6, we look briefly at some aspects of plant development, which differs in some respects from that of animals but involves similar principles. The development of individual organs such as the vertebrate limb, the insect eye, and the nervous system illustrates multicellular organLation and tissue differentiation at later stagesin embryogenesis,andwe consider someof these systemsin deail in Chapters 8-10. We also deal with the development of sexual draracteristics (Chapter 11). The study of developmental biology, however, goes well beyond the development of the embryo. We also need to understand how some animals can regenerate lost organs (Fig. 1.2, and Chapter 13), and how post-embryonic growth of the organism is contrclled, a process that includes metamorphosis and aging (Chapter 12). Taking a wider view, we consider in Chapter 14 how developmenal mechanismshave evolvedand how they constrain the very processof evolution itself, One might ask whether it is necessary to cover different organisms and developmental systems in order to understand the basic features of development. The answer at present is yes. Developmental biologists do indeed believe that there are general principles of development that apply to all animals, but that life is too wonderfi,rlly diverse to find all the answers in a single organism. As it is, develop mental biologists have tended to focus their efforts on a relatively small number of animals, chosen originally becausethey were convenient to study and amenable to experimental manipulation or genetic analysis.This is why some creatures, such as the frog Xenopu laais (Fig. 1.3), the nematode Caenorlwbditis,and the fruit fly Drosophila,have such a dominant place in developmental biology, and are encountered again and again in this book. Indeed, it is very encouraging that so few systemsneed to be studied in order to understand animal development. Similarly, the thale-cress,Arabiilopsisthahana,can be used as a model plant to consider the basic features of plant development. One of the most exciting and satis$ring aspectsof developmental biology is that understanding a developmental process in one organism can help to illuminate similar processeselsewhere,for example in organismsmuch more like ourselves. Nothing illustrates this more dramatically than the influence tltat our understanding of Drosophiladevelopment, and especially of its genetic basis, has had throughout developmental biology. In particular, the identification of genes controlling early embryogenesis in Drosophilahas led to the discovery of related genes being used in similar ways in the development of mammals and other vertebrates. Such discoveries encourageus to believe in the emergenceof general developmental principles. Frogs have long been a favorite organism for studying development because their eggsare large, and their embryos are robust, easyto grow in a simple culture medium, and relatively easy to experiment on. The South African frog Xenop.tsis the model organism for many aspects of vertebrate development, and the main features of its development (Box 1A, pp. 4-5) serve to illustrate some of the basic stages of development in all animals. The early development of Xenopw and the other model vertebratesis discussedin Chapters3 and 4. In the rest of this chapter we first look at the history of embryology-as the study of developmental biology has been called for most of its existence. The term BL IOLOC Y TH E OR IC IN SOF D E V E LOP M E N TA f,l of ffy ti s io f tnt rat to f EF, developrnental biology itself is of much more recent origin. We then introduce somekey conceptsthat are used repeatedly in studying and understanding develop ment. biology Theoriginsof developmental Many questions in embryology were first posed hundreds, and in some cases thousands,ofyears ago.Appreciating the history ofthese ideas helps us to understand why we approach developmental problems in the way that we do today. fi& rail tics md GII rrh rnc rrtal relf. md ENL tere itoo lop lo f hto ta s tf ly nrnfew lrly' : the tbat nate lves. di:rg hout Early used tcovples. zuse lure rls is nain besic il the r tle tern r.r Aristotle first defined the problem of epigenesisand preformation A scientific approach to explaining development started wittt HipPocratesin Greece in the Sth century nc. Using the ideas current at the time, he tried to explain developmentin terms of the principles of heat, wetness,and solidification. About a century later the study of embryology advanced when the Greek philosopher Aristotle formulated a question that was to dominate much thinking about development until the end of the 19th century.Aristotle addressedthe problem of how the different parts of the embryo were formed. He considered two possibilities: one was that everything in the embryo was preformed from the very beginning and 5imply got bigger during development; the other was that new structures arose progressively,a processhe termed epigenesis(which means 'uPon formation') and that he likened metaphorically to the 'knitting of a net'. Aristotle favored epigenesis and his conjecturewas correct. Aristotle's influence on Europeanthought was enounous and his ideasremained dominant well into the 17th century. The conffary view to epigenesis,namely that the embryo was preformed from the beginning, was championed anew in the late 17th century. Many could not believe that physical or chemical forces could mold a living entity like the embryo. Along with the contemPoraneous background of belief in the divine creation of the world and all living things, was the belief that all embryos had existed from the beginning of the world, and that the first embryo of a speciesmust contain all future embryos. Even the brilliant 17th century Italian embryologist Marcello Malpighi could not free himself from preformationist ideas.While he provided a remarkably accurate description of the development of the chick embryo, he remained convinced, against the evidence of his own observations, that the embryo was already present from the verybeginning (Fig.1.4).He arguedthat atvery early stagesthe parts were so small that they could not be observed,even with his best microscope.Yet other preformationists believed that the sperm contained the embryo and some even claimed to be able to see a tiny human-an homunculus-in the head of each human sperm (Fig.1.5). The preformation/epigenesis issue was the subject ofvigorous debate throughout the 18th century. But the problem could not be resolved until one of the great advancesin biology had taken place-the recognition that living things, including embryos,were composedof cells. made Malpighi's drawings, Thefigureshows of thechickembryo. description Fig.1.4 Malpighi's (bottom). Hisdrawings accurately (top),andat2 days' incubation theearlyembryo in1673, depicting oftheembryo. andbloodsupply illustrate theshape Reprintedby permissionof the Presidentond Councilof the RoyolSociety. E Fig. 1.3 Photographofthe adult South Africancfaw-toedfrog, )knopus loevis. Scalebar= 1 cm. courtesy ofl. Smith. Photogroph 3 4 t: Htsro RyANDBAs t cc oNc Epr s BoxlA Basicstages of Xenopus |oevisdevetopment @& Egg *ffir{eee %u:* @ (section) Gastrula Vegeratpole / / ("/ ^& Fettilization Gastrulation Metamorphosis Free-swimming tadpole ftrture | 5ta 9e12I Al (section) venrlar t(. 0rganogenesis \\ \\ Antedor \\==- '*--4ffii ;'J'#ff/ """' ll&i{ somiteslffii'|j ,f^l / "' 1 u* un) iooo.r {w t\l -\p rorkmass \- Posteriol Tailbud-sta9e embrlo (dorsalviewwith surfareremoved) ==-f / Tailbud*tage embryo (latentiiew) Antedor ffi neural folds Neurula I Stage13| (surface removed to reveal notochord) Postelior @ (dorsal view) @ C oni --. IH E O R IGIN S O F D EVELOPM ENTA L B I O L O G Y Box 1A (continuedl Basic stages of Xenopusloevisdevelopment Although vertebrate development is very varied, there are a number of basic stages that can be illustrated by following the developmentof the frog Xenopusloevis,which is a favorite ectoderm, which forms both the epidermisand the nervous system. The future endoderm and mesoderm, which are destinedto form internalorgans,are still on the surfaceof the organism for experimentalembryology.The unfertilizedegg is a largecell.lt hasa pigmenteduppersurface(the animal pole) embryo. During the next stage-gastrulation-there is a dramatic rearrangementof cells;the endodermand mesoderm move inside,and the basicbody plan of the tadpole is estab- and a lower region (the vegetal pole) characterizedby an of yolkgranules.Soevenat the beginning,the egg is accumulation cellsfrom the animalhalf development, not uniform;in subsequent becomethe anterior(head)endof the embryo. After fertilizationof the egg by a sperm,and the fusionof male and female nuclei,cleavage begins.Cleavagesare mitotic divisionsin which cellsdo not grow betweeneachdivision,and so cleavagesthe cellsbecomesmaller.After about with successive 12 divisioncycles,the embryo,now knownasa blastula,consists of many small cellssurroundinga fluid-filledcavity (the blastocoel) abovethe largeryolky cells.Already,changeshaveoccurred within the cellsand they haveinteractedwith eachother so that some future tissue types-the germ layers-have become partly specified.The future mesoderm for example,which gives riseto muscle,cartilage,bone,and otherinternalorganslikethe heart,blood,and kidney,is presentin the blastulaasan equatorial band.Adjacentto it is the future endoderm, which givesrise to the gut, lungs,and liver.The animalregionwill give riseto lished.Internally,the mesodermgivesriseto a rod-likestructure (the notochord), which runs from the headto the tail, and lies centrallybeneaththe future nervoussystem.On either side of the notochord are segmented blocks of mesoderm called somites, which will give rise to the musclesand vertebral column.aswellasthe dermisof the skin. Shortly after gastrulation,the ectoderm above the notochord foldsto form a tube (the neural tube), which givesriseto the brain and spinalcord-a processknown as neurulation. By this time, other organs,suchas limbs,eyes,and gills,are specifiedat their future locations,but onlydevelopa little later,duringorganogenesis. cellssuch as muscle,cartilage, specialized Duringorganogenesis, Within48 hoursthe embryohasbecome and neuronsdifferentiate. a feeding tadpole with typical vertebrate features. Becausethe conditiming of eachstagecanvary,dependingon environmental andotherembryosareoften stagesin Xenopus tions,developmental denotedby stagenumbersratherthan by hoursof development. r.z Celltheory changedthe conceptionof embryonic development and heredity The cell theory developedbetween 1820 and 1880 by' among others, the German botanist Matthias Schleidenand the physiologistTheodor Schwann,was one of the most illuminating advancesin biology, and had an enorrnousimpact. It was at last recognizedthat all living organisms consist of cells, which are the basic units of life, and which ariseonly by division fiom other cells.Multicellular organismssuch as animals and plants could now be viewed as communities of cells. Development could not therefore be based on preformation but must be epigenetic, because during development many new cells are generatedby division fiom the egg, and new tj4)es of cells are formed. A crucial step forward in understanding development was the recognition in the 1840sthat the egg itself is but a single, albeit cell. specialized, An important advancewas the proposal by the 19th century German biologist August Weismann that the offspring does not inherit its characteristicsfrom the body (the soma)of the parent but only from the germ cells-egg and sperm-and that the germ cells are not influenced by the body that bears them. weismann thus drew a fundamental distinction between germ cells and body cells or wascurledupin theheadof each thatanhomunculus believed Fig.1.5 Somepreformationists sPerm, (1694). Hortsoeker An imoginotivedrowing,ofter Nicholos r 6 l: HtsroRyANDBAstccoNcEprs | | mutationsin ||;:T;#:.f1t,f,,,. /a<l Et r E ll Fig. 1.6 Thedistinction between germ cells and somaticcells.In eachgenerationgermcells giveriseto both somaticcellsandgermcells, but inheritance isthroughthe germcellsonly. Changes that occurdueto mutationin somatic cellscanbe passed onto theirdaughtercellsbut do not affectthe germline. Fll ll ll o-ol{{ zlgote .+ germcel l s j- o- , = / n . . lE / /t' l a /d € < ) o -: _ o-o-|t{=o-oll{ L-------___a__| | zygote germcel l s ll | | zygote L_______________ germcel k l l '- somatic cells(Fig.1.6).characteristicsacquired by the body during an animal's life cannot be transmitted to the germline. As far as heredity is concerned, the body is merely a carrier of germ cells. As the English novelist and essayist samuel Butler put ic "A hen is only an egg's way of making another egg.,' work on sea urchin eggs showed that after fertilization the egg contains two nuclei, which eventually fuse; one of these nuclei belongs to the egg while the other comes from the sperm. Fertilization therefore results in an egg carrying a nucleus with contributions from both parents, and it was concluded that the cell nucleus must contain the physical basis of heredity. The climax of this line of research was the eventual demonstration, toward the end of the 19th century, that the chromosomes within the nucleus of the fertilized egg-the zygote-are derived in equal numbers from the two parental nuclei, and the recognition that this provided a physical basis for the transmission ofgenetic characters according to laws developedby the Austrian botanist and monk Gregor Mendel. The constancy of chrornosome number from generation to generation in the somatic cells was found to be maintained by a reduction division (meiosis) that halved the chromosome number in the germ cells, whereas somatic cells divide by the processof mitosis, which maintains chromosome number. The precursors to the germ cells contain two copies of each chromosome, one maternal and one paternal, and are called diploid. This number is halved by meiosis during formation of the gametes, so that each germ cell contains only one copy of each chromosome and are called haploid. The diploid number is restored at fertilization. r.3 Two main types of development were originally proposed once it was recognized that the cells of the embryo arose by cell division from the zygote, the question emerged of how cells becarne different from one another. with the increasing emphasison the role of the nucleus, in the 1gg0sweismann put forward a model of development in which the nucleus of the zygote contained a number of specialfactors or determinants (Fig. 7.7).He proposedthat while the fertilized egg underwent the rapid cycles ofcell division known as cleavage,these determinants would be distributed unequally to the daughter cells and so would control the cells' future development. The fate of each cell was therefore predetermined in the egg by the factors it would receive during cleavage. This type of model was termed fmosaic', as the egg could be consideredto be a mosaic of B IIOLOGY TH E OR IC IN SOF D E V E LOP MEN TA I-l cl "l Flg. 1.7 Weismann'stheory of nuclear determination.Weismannassumedthat there werefactorsin the nucleusthat weredistributed to daughtercellsduring asymmetrically cleavageanddirectedtheirfuturedevelopment. EI I ,l __t life ly is der trro the Fng :cell eof Ery' -are that ilirg |ncy was lne rs of rells il are ttes, rlled rttre ther. n;rnn dned e the lhese Fuld rede i tyPe rk of rr 7 discretelocalized determinants.Central to Weismann'stheory was the assumption tbat early cell divisions must make the daughter cells quite different from each other as a result ofunequal distribution ofnuclear components. In the late 1880s,initial support for Weismann's ideas came from experiments carried out independently by the German embryologist Wilhelm Roux, who experimented with frog embryos. Having allowed the first cleavage of a fertilized frog eg, Roux destroyed one of the two cells with a hot needle and found that the remaining cell developed into a well-formed half-larva (Fig. 1.8).He concluded that the 'development of the frog is based on a mosaic mechanism, the cells having rheir character and fate determined at each cleavage". But when Roux's fellow countqrman, Hans Driesch,repeatedthe experiment on seaurchin eggs,he obtained quite a different result (Fig.1.9).He wrote later: 'But things turned out as they were bound to do and not as I expected; there was, typically, a whole gastrula on my dish the next morning, differing only by its small size from a normal one; and this small but whole gastrula developed into a whole md typical lawa." Driesch had completely separated the cells at the two-cell stage and obtained a normal but small larva. That was just the opposite of Roux's result, and was the first clear demonstration of the developmentalprocessknown as regulation. This is the ability of the embryo to develop normally even when some portions are renoved or rearranged. We shall see many examples of regulation throughout the book (an explanation of Roux's experiment, and why he got the result he did, is also given later, in Section3.7). r.l The discoveryof induction showed that one group of cellscould determine the developmentof neighboring cells Ahhough the concept of regulation implied that cells must interact with each other, fre central importance of cell-cell interactions in embryonic development was not really establisheduntil the discovery of the phenomenon of induction, in which me cell, or tissue, directs the development of another, neighboring, cell or tissue. The importance of induction and other cell-cell interactions in developmentwas pmved dramatically in7924when Hans Spemannand his assistantHilde Mangold wried out a famous transplant experiment in amphibian ernbryos. They showed &at a partial second embryo could be induced by grafting one small region of m early newt embryo onto another at the same stage (Fig. 1.10).The grafted tissue was taken from the dorsal lip of the blastopore-the slit-like invagination frat forms where gastrulation begins on the dorsal surface of the amphibian rmbryo (seeBox 1,4.,pp. 4-5). This small region they called the organizer, since it seemedto be ultimately responsible for controlling the organization of a complete ;lfr 8 t: HtsroRYANDBAstccoNcEPTs Fig. 1.8 Roux'sexperimentto investigate Weismann'stheory of mosaicdevelopment. Afterthe first cleavageof a fiog embryo,oneof thetwo cellsiskilledby prickingit with a hot needle; the otherremains undamaged. At the blastula stagethe undamaged cellcanbeseen to havedividedasnormalintomanycellsthatfill halfoftheembryo.Thedevelopment ofthe blastocoel is alsorestrictedto the undamaged half.Inthe damagedhalfof the embryo,no cells appearto haveformed.At the neurulastage, the undamaged cellhasdeveloped into something resembling halfa normalembryo. Fig. 1.9 Theoutcomeof Driesch's experimenton seaurchin embryos,which first demonstratedthe phenomenonof regulation.Afterseparationof cellsat the twocellstage,the remainingcelldevelopsinto a small,butwhole,normallarva.Thiscontradicts Roux's earlierfindingthat if oneofthe cellsof a two-cellfrogembryoisdamaged, the remaining celldevelops intoa half-embryo only(seeFig.1.8). embryonic body, and it is commonly known as the Spemann-Mangoldorganizer, or just the Spemannorganizer. For their discovery,Spemannreceived the Nobel Prize for Physiology or Medicine in 1935, one of only two ever given for embryological research. Sadly, Hilde Mangold had died earlier, in an accident, and so could not be honored. 1.5 The study of developmentwas stimulated by the coming together of geneticsand development During much of the early part of the twentieth century there was little connection between embryolory and genetics.When Mendel'slaws were rediscoveredin 1900 there was a great surge of interest in mechanisms of inheritance, particularly in relation to evolution, but less so in relation to development.Geneticswas seenas the study of the transmission of hereditary elements from generation to generation, whereas embryolory was the study of how an individual organism develops and, in particular, how cells in the early embryo became different from each other. Geneticsseemed,in this respect,to be irrelevant to development. BL IOLOGY TH E OR IGIN SOF D E V E LOP ME NTA graftedfinman lip of blastopore andMangoldof inductionof a newmain Dorsal bySpemann Fig.1.10 Thedramaticdemonstration of newtto the spedes from unoicrirented A pieceoftissue(yellow) gastrula. amphibian eariy in the region spe<ies bodyaxisbytheorganizer blasioioelroofof a pigmented a of side opposite to the grafted is gastrula a newt(Iritoncristotus) thedorsallipoftheblastopore-of gastrufafromanother,pigmented,newtspecies(Tritontoeniotus'pink)'Thegraftedtissueinducesa newbodyaxiscontainingneura|tubeandsomites.Theunpigmentedgrafttissueformsanotochord atitsnewsite(seesectionin|owerpane|)buttheneura|tubeandtheotherstructuresofthenewaxis and byspemann discovered region Theorganizer hosttissue. fromthepigmented havebeeninduced organizer' isknownastheSpemann Mangold to link genetics and embryoloey An important concePt'that eventually helped phenotype. This was first put forward was the distinction between genotype and in 1909' The genetic endowment of an by the Danish botanist Wilh"l* ;oh"tt"'"n r, EI lgi ild rcn 900 7in Das Eralops fur. organism_thegeneticinformationitacquiresfromitsparents_isthegenotype' ItsvisibleaPpearance,internalstructufe,andbiochemistD/atanystageofdevelopmentisthephenotyPe.WhilethegenotypecertainlycontrolsdeveloPment'envigenotype influence the phenotype' Despite ronmental factors interacting with the considerable differences in identical genogpes, identical twins can develop having-phenotypes tend to become more as ttrey grow up (Fig' 1'11)' and these ttreir posed in terms of the be now could evident with age.The problem of development genetic endowment the how relationship between genotyPe and phenotype; give rise to a functioning to during development becomes'ffanslated' o,;"*p'"""a' organism. was a slow and tortuous The coming together of genetics and embryology and function of the genes process. Little progress was made until the nature in the 1940s that genes encode were much better understood' The discovery proteinswasamajorflmingpoint.Asitwasalreadyclealthatthepropertiesofa the fundamental role of genes in cell are determined Uy tne f,roteins it contains' developmentcouldatlastbeappreciated.BycontrollingwhichProteinswere in cell proPerties and behavior made in a ceII, genes could control the changes (induced) embrYo setondary thatoccurredduringdevelopment.Afurthermajoradvanceinthelg60swasthe understandingthatsomeg"n"s",,cod"proteinsthatcontroltheactivityofother genes. r--l rr €= I FI F E r.6Deve|opmentisstudiedmain|ythroughaselectionofmode|organisms Althoughthedevelopmentofawidevarietyofspecieshasbeensttrdiedatonetime oranother,arelativelysmallnumberoforganismsprovidemostofourknowledge thus regard them as models for about developmental mechanisms' We can understandingtheprocessesinvolvedandtheyareoftencalledmode|organisms. Seaurchinsandamphibianswerethemainanimalsusedforthefirstexperimentalinvestigationsatthebeginningofthetwentiethcenturybecausetheirdevelop. large obtain and' in the caseof the frog' sufficiently ing embryos are both ""'y1o androbustforrelatively"",y"*p",i*entalmanipulation,evenatquitelatestages' (Ws mtsathrs)'the chicken (Gallus Among vertebrates, th; fro; X'bevis' the mouse gollus),andthezebrafish(Dantoreriol'arethemainmodelorganismsnowstudied' .{monginvertebrates,thefruitflyD.me|anogasterandthenematodeworrnC.e|egans a great deal is known about their have been the focus ofmost attention, because easily genetically modified' With the developmental genetics and they can also be adventofmodernmethodsofgeneticanalysis,therehasalsobeenaresurgenceof For plant developmental interest in the sea urchin stronglocentrotuswluratus. organism' The life model main the as hiology, the thale-cress L thakann serves Fig. 1.11 Thedifferencebetween genotype and phenotype.Theseidenticaltwins havethe onefertilizedeggsplit samegenotypebecause Theirslight development' during two into isdueto nongenetic in appearance difference influences' factors,suchasenvironmental of JosiondloimePoscuol' courtesy Photogroph 10 A N D B A 5 ICCONCEPT 5 1: H I S T O R Y cyclesandbackgtounddetailsforthesemodelorganismsaregivenintherelevant chapters later in the book. firereasonsforthesechoicesarepartlyhistorical-onceacertainamountof Iesearchhasbeendoneononeanimalitismoreefficienttocontinuetostttdy with another species-and partly a it rather than start at the beginning again Each specieshas its advantages question of ease of study and biotogicat i"t:tIt anddisadvantagesas"aet'etopment"tmodel'Thechickembryo'forexample'has eggsare of vertebrate developmentbecause fertile longbeen studied as * "*"*ft" experimental microsurgical manipulation easily available, the embryo -i'tt"*a' egg' A disadvantage' however' was very well, and it can be cultured outside the genetics.Howevet we know that little was known about the chick's devel0pmental the mouse is more difficult although about the genetics of the mouse' ;;;;"; tostudyinsomewaysasdevelopmenttakesplaceentirelywithinthemother. Manydevelopmentalmutationshavebeenidentifiedintfiemouse,anditisalso techniques' It is also the best amenable to genetic modification by transgenic mammalian development' including experimental model we have for studying th a to fh u n ans.Thezebrafi shi samorerecentaddi ti ontothesel ectl i stof are in large numbers' the embryos vertebrate model systems; it is easy to breed transparentandsocelldivisionsandtissuemovementscanbefollowedvisually, and it has great potential for genetic investigations' to understand how genes control A major goal of developniental biology is embryonicdevelopment,andtodothisonemustfirstidentiffthosegenescritiThis task can be approached in a variety cally involved in controlling development' ofways,dependingonthe"organisminvolved,butthegeneralstaltingpointistlre identificationofmutationsthatalterdevelopmentinsomespecificandinformativeway,asdescribedinthefollowingsection;Techniquesforidentiffinggenes manipulating their expression in the that control development and detecting and along with techniques for genetic organism are described throughout the book' maniPulation. S o m e o f ourmodel organi smsaremoreamenabl etoconventi onal genet ic in developmental biology' little analysis than others. Despite its importance has the disadvantage of conventional genetics has teen done on Xlnwis'which atetrap|oidgenome(onewithfoursetsofchromosomesinthesomaticcells)and years to reach sexual maturity. Using a relatively long breeding period, taking 1-2 however' many developmental genes modern genetic ana uioiiformatics methods' sequencecomparison with known have been identifled ir_X'lastisby direct DNA genesinorganismssuchasDrosoptn|nandmice.ThecloselyrelatedfrogXenoptls (Silurana)troptcaltsisamuchmoreattractiveorganismforgeneticanalysis;itis to produce ffansgenic organisms' diploid and can also be genetically manipulated identify develop is now being sequenced'which will help T:heX. troprtcalisgenome mentalgenesinX.laevis.Thesituationwithbirdshasbeenrathersimilar'buthere can be used to identiff develalso, direct DNA comparisons with other organisms the chicken genome has now been oPmental g"n"s. tt e DNA sequence of completed,whichshouldbeaconsiderablehelpinthegeneticanalysisofdevelop mentinthisspecies.Sequencingofthegenomeoft}reseaurchinS'plpwaws, biology, is also under way' one of the oldest model organirms i" developmental Completegenomer"qo""t"'ht""beenavailableforsomeyearsforhuman'mouse' Drosoplila, and Caensrlwb ditis' gene tral been identified in In general, when an important developmental consider whether a corresponding one animal, it has proved iery ruw"rdirrg to BL IOLOGY TH EOR IGIN SOF D E V E LOP ME N TA EC gene is present and is acting in a developmental capacity in other animals. Such genes are often identified by a sufficient degree of nucleotide sequencesimilarity to indicate descent from a conrmon ancestral gene. Genes that meet this criterion are known as homologous genes. As we shall see in Chapter 4, this approach identified a hitherto unsusPected class ofvertebrate genes that speciff the regular segmented pattern from head to tail that is represented by the different grpes of vertebrae at different positions. These genes were identified by their homolory with genes that speciff the identities of the different body DII segments in Drosoplfila. trt of ty 'a Fs Ets 11 a5 tr/ Elt Ef. bo ESt ng of EC [y, rol itiEty he nirDes fhe ctic rtic ttle :o f nd ing nes rvn |p{ls r.z The first developmentalgeneswere identified as sPontaneousmutations Most of the organisms dealt with in this book are sexually reproducing diploids: their somatic cells contain two copies of each gene, with the exception of those on sex chromosomes.X. lawis is an exception, as it is tetraploid; that is' it has four copies of the basic genome in its somatic cells, and two copies in the germ cells. This means that it will have up to four copies of each gene in its somatic cells, which complicatesgenetic analysis.In a diploid sPecies,one copy,or allele, of each gene is contributed by the male parent and the other by the female. For many genes there are several different 'normal' alleles present in the poPulation, which leads to the variation in normal phenotype one seeswithin any sexually reproducing species.Occasionally,however, a mutation will occur spontaneously in a gene and there will be marked change, usually deleterious, in the phenotype of the organism. Many ofthe genes that affect development have been identified by spontaneous mutations that disrupt their function and Produce an abnormal phenotype. Mutations are classifiedbroadly according to whether they are dominant or recessive (Fig. 1.12). Dominant and semi-dominant mutations are those that produce a distinctive phenotype when the mutation is present in just one of the alleles of mutation(e.9.wfiite-) Recesive Genotype Phedotype Genotype Pheno0pe wildtype white+ normal wildtype nomal tis w{K llrs. j@x. bq white+ Ere :ve1- fiIs, r:ry. nse, r nomal \ .e,X;ffi; @K white- mutation homozygous whitt ili n iling re \, mutation heterozygous Ben bp mutation(e.9.Bnchryryl Semi-dominant x!, x@{. whitt ir re f/\ eyes white @ + X}@(K i*@& + mutation heterozygous e@e r"sGWX "d{-\ '*S*+*-t ,....*,***,1, tail deformed \*fu+s ",il*^\ T mutation homozygous T xs@{K X@K T lethal embryonic {,& Fig.1.12 Typesof mutations.Left:a mutation whenit onlyhasaneffectin the is recessive state;that is,whenboth copiesof homozygous the genecarrythe mutation.Right:by contrast, mutation a dominantor semi-dominant in the produces aneffecton the phenotype state;that is,whenjust onecopy heterozygous A plussign of the mutantgeneispresent. denoteswildtype,anda minussignrecessive. f isthe mutantform of the geneBrochyury' t2 1 : HIS T ORY A N D BASIC CONCEPTS a pair; that is, it exerts an effect in the heterorygous state. Dominant mutations can be missed as they are often lethal. By contrast, recessive mutations such as white in Drosopli)a,which produces flies with white eyes rather than the normal red, alter the phenotype only when both alleles of a pair carry the mutation; that is, when they are homozygous. In general, dominant mutations are more easily recognized, particularly if they affect gross anatomy or coloration, provided that they do not causethe early deati of the embryo in the heterozygous state. Truly dominant mutations are rare, however. A mutation in the mouse gene Braclryuryis a classic example of a semidominant mutation and was originally identified because mice heterozygous for this mutation (symbolized by Q have short tails. When the mutation is homozygous it has a much greater effect, and embryos die at an early stage, indicating that the gene is required for normal embryonic development (Fig.1.13).Oncebreeding studies had confirmed that the Brachyny ffait was due to a single gene, the gene could be mapped to a location on a particular chromosome by classical genetic mapping techniques. IdentiSring recessive mutations is more laborious, as the heterozygote has a phenotype identical to a normal wild-type animal, and a carefully worked-out breeding program is required to obtain homozygotes. Identiffing potentially lethal recessivedevelopmental mutations requires careful observation and analysis in mammals, asthe homozygotesmaydie unnoticed inside the mother. Very rigorous criteria must be applied to identit/ those mutations that are affecting a genuine developmental process and not just affecting some vital but routine housekeeping function without which the animal cannot suryive. One simple criterion for a developmental mutation is embryonic lethality, but this also catches mutations in genes involved in housekeeping functions. Mutations that produce abnormal patterns of embryonic development are much more promising candidates for true developmental mutations. In later chapters we shall see how large-scale screening for mutations following mutagenesis using chemicals or X-rays has identified many more developmental genes than would ever have been picked up by rare spontaneous mutations. & o @. -:.=*.-/., Shorttail ;1 Fig. 1.13 Geneticsof the semi-dominant mutation Brcchyury(I)in the mouse.A male heterozygote carryingthe f mutationmerely hasa shorttail.Whenmatedwith a normal (wildtype,++)female,someof the offspringwill alsobe heterozygotes andhaveshorttails. Matingtwo heterozygotes togetherwill resultin someofthe offspringbeinghomozygous (I/f for the mutation,resulting in a severe andlethal developmental abnormality in whichthe posteriormesodermdoesnot develop. r H@,@dre\ =-'5)v,.-:+*ffi iffi@@ ffiifiHy,ii;iits., K IT A C ON C E P TUATOOL L I 5 I t v h Ii 5ummary startedwith the Greeksmorethan 2000years Thestudvof embryonicdevelopment not containedcomp|ete|y ,oo' n'i'totru put forwardthe ideathat embryoswere emergedgradually' structure and form that but egg, ,ne *',n,^ ;eformed"in by "i;.irr" in the 17thand18thcenturies Thisideawaschallenged developed. .ri6u orever "tUruo had been, that embryos at thit idea il;;ijJ,"*i',rior"t"rn.'roon,,r," of the cell The emergence *""fa i", had existedfrom the beginningof the world' t heor v int h e l g th c e n tu ry fi n a | | y s e tt| e dthei ssuei nfavorofepi genesi s' andi tw as someofthe cells' specialized, hishly albeit lss;'u ''"gr", to regutate, able are embryos urchin ,io*"4 iirt u"ryu.1ysea the established rhis killed' or it."ttr.r" '"mou.d "u"n communication on in least at mustdepend Part tiril"u"ropment rg :";rJilffi;;;; .#;;;;*"-r,t, il;;il;;;;;iv ;;;;il;..il" [e #;";; dc fi",#;;;il;;;;;;."r* ;;f ;;ilil;;;; }I vet lle [y ng on er. of cell-cell 6," ."it, of the embryo.Direct evidencefor the importance and outbvspemann carried n'.n experiment could resion orsanizer ,r,-.,,r,".-"rr,or ihe amphibian embrvo' intoanother whentransplanted ;;;;;;;; ;tiai"murv-otromhosttissue inthe hasonlybeenfullyappreciated development genes incontrolling ii" J" much made "rir,i.iJii"'rr"lr hasbeen of development the-seneticbasis ;;;,;;; "ftechniques biology' of molecular timesbythe in recent easier rrbe Ple [es tlce tres ale bas lu p A conceptualtoolkit complicated fate a single Development into a multicellular organism is the most and the challenge of living cell can undergo; in this lies both the fascination developmentalbiology.Yetonlyafewbasicprinciplesareneededtostafttomake ,"rrr"ofdevelopmentalprocesses.Therestofthischapterisdevotedtointroducrepeatedly throughout ing these key concepts.These principles afe encountered systems' and developmental and organisms the book, as we look at different embarking on a study of should be regardedas a conceptualtool kit, essentialfor development. Genescontroldevelopmentbycontrollingwhereandwhenproteinsaresyntheactivity setsup intracellusized,and many thousandsof genesare involved. Gene genes' and between proteins and proteins lar networks of interactions between one ofthese ProPerand proteins, that confer on cells their particular Propefties. in specifiedways' cells other to ties is the ability to communicatewith and respond it is t hes ece | | -c e l l i n te ra c ti o n s th a td e t ermi nehow theembryodevel ops;no developmentalprocesscanthereforebeattributedtothefunctionofasinglegene information on developor single protein. The amount of genetic and molecular mentalProcessesisnowenolmous.Inthisbook,wewillbehighlyselectiveand into the mechanismsof describeonly those molecular details that give an insight developmentand illustrate general principles' of pattern' t.E Developmentinvolvescell division,the emergence changein form, cell differentiation, and growth structules from an initiauy Developmentis essentiallythe emergenceof organized r,erysimplegroupofcells.Itisconvenienttodistinguishfivemaindevelopmental influence one another pr*.rr"r, even though in reality they overlap with and considerablY. II 13 14 1 : HIS T ORY A N D B ASIC CONCEPTS Fig.1.14 LightmicrographofXenopus eggs afterfour celldivisions.Scalebar= 1 mm. Photogroph courtesy ofl. Slock. The first is the processknown as cleavage,which is the division of the fertilized egg into a number of smaller cells (Fig. 1.14).Unlike the cell divisions that take place during cell proliferation and growth of a tissue, there is no increasein cell massbetween eachcleavagedivision. The cell rycles during cleavageconsistsimply of phases of DNA replication, mitosis, and cell division, with no intervening stageof cell growth. We shall discussthe various types of cell rycle in Chapter 12. The cleavage stage of embryogenesis thus rapidly divides the embryo into a number of cells, each containing a copy of the genome. Patternformation is the processbywhich a spatial and temporal pattern of cellular activities is organized within the embryo so that a well ordered structure develops. In the developing arm, for example, pattern formation is the processthat enablescells to 'know' whether to make an upper arm or fingers, and where the muscles should form. There is no single universal strategy or mechanism of patterning; rather, it is achieved by a variety of cellular and molecular rnechanisms in different organismsand at different stagesof development. Pattern formation initially involves laying down the overall body plan-defining the main body axes of the embryo so that the head (anterior) and tail (posterior) ends, and the back (dorsal) and underside (ventral) are specified. Most of the animals we shall deal with in this book have a head at one end and a tail at the other, with the left and right sidesof the body being bilaterally synrmetrical-that is, a mirror image of each other. In such animals, the main body axis is the anteroposterior axis, which runs from the head to the tail. Bilaterally s5rmmetrical animals also have a dorso-ventral axis, running fiom the back to the belly. A striking feature of these axes is that they are almost always at right angles to one another and can thus be thought ofas making up a systemofcoordinates on which any position in the body could be specified(Fig.1.15).In plants, the main body axis runs from the growing tip (the apex)to the roots and is known as the apical-basal axis. Plants also have radial symmetry with a radial axis running from the center of the stem outwards. Even before the body axesbecome clear, eggsand embryos often show a distinct polarity, which in this context means that one end is distinguishable from the other in its structure or properties. Many individual cells in the developing embryo also have polarity, with one end of the cell structurally or functionally different from the other. The next stagein pattern formation in animal embryos is allocation of cells to the different germ layers-the ectoderm,mesoderm,and endoderm (Box 18, p. 15). During further pattern formation, cells of these germ layers acquire different 0@ lM iflw nMl dr I j$@ m Itr geembryo ,Y.laevirtailbud.sta @ il!M' (back) Donal rffi0 ,u itE th ,: Anterior (head) Posterior (tail) rym Fig.1.15 Themainaxesof a developing embryo.Theantero-posterior axisandthe dorso-ventral axisareat rightanglesto one another, asin a coordinate svstem. m g& ,tN (front) Ventral im@ ilh A C ON C EPT U AL T O O L K I T Box 1B Germ laYers The concept of germ layers is useful to distinguish between regions of the early embryo that give rise to quite distinct types of tissues.lt appliesto both vertebratesand All the animalsconsideredin invertebrates. .c ri -': this book, except for the coelenterateHydro' are triploblasts,with three germ layers:the endoderm,which givesriseto the gut and its derivatives,suchasthe liverand lungsin vertebrates:the mesoderm,whichgivesriseto the skeleto-muscularsystem, connectivetissues' and other internalorganssuch as the kidney and heart; and the ectoderm,which givesrise to the epidermisand nervoussystem'These The boundearlyin development' arespecified befuzzy can layers different ariesbetweenthe neural The exceptions' and there are notable crestin vertebrates,for example,is ectodermal in origin but gives rise both to neuraltissue and to some skeletalelements,which would in origin' mesodermal normallybe considered -jentitiessothatorganizedspatialpattelnsofcelldifferentiationemerge,suchas and the -re arrangement of skin, muscle, and cartilage in developing limbs' In the earliest stagesof pattern _-rangementof neurons in the nervous system. ::rmation,differencesbetweencellsarenoteasilydetectedandprobablyconsist changein activity of a very few genes' -: subtle diffbrencescausedby a process is change in form' or The third important developmental changesin three-dimenrorphogenesis (Chapter7). Embryosundergo remarkable .lonalform_youneedonlylookatyoufhandsandfeet.Atcertainstagesindevelchanges in form' of which , lment, there are characteristic and dramatic undergo gastrulation' embryos all animal ; astrulatlon is the most striking. Almost During gastrulaplan emerges. ::ring which the gut is formed and the main body such as the animals in move inwards and' -rn, cells on the outside of the embryo into blastula hollow spherical ,.a urchin, gastrulation even transforms a in Morphogenesis gut (Fig. 1.16). . gastrulawith a hole through the middle-the of cells the of Most embryos can also involve extensive cell migration' tissue -rrmal a from -:e human face, fbr example, are derived from cells that migrated on the back of the embryo' --Lledthe neural crest' which originates Thefourthdevelopmentalprocesswemustconsiderhereisecl|differentiation,in .hjchcellsbecome,.*.*,"uyandfunctionallydifferentfromeachother,ending is a as blood' muscle' or skin cells' Differentiation -; as distinct cell types such =adualplocess,cellsoftengoingthroughseveraldivisionsbetweenthetimeat differentiated (when .'nich they start differentiating and the time they are fully ,-necelltypesstopdividingaltogether)'andisdiscussedinChapter8'Inhumans' least 250 clearly distinguishabletypes ofcell. ---:efertilized egg gives rise io "t r lnsects l5 16 . 1: HlsroRYANDBAslccoNcEPTs Fig. 1.16 Gastrulationin the seaurchin' GJrulation transformsthe sphericalblastula into a structurewith a holethroughthe middle' has the gut. Theleft-handsideof the embryo beenremoved. w#M# Patternformationandcelldifferentiationarevelycloselyinterrelated,aswe canseebyconsideringthedifferencebetweenhumanarmsandlegs.Bothcontain tle cartilage' bone' skin' and so on-yet exactly the same tyPes of cel-muscle' different' It is essentially pattem pattern in which they are arranged is clearly elephants and chimpanzees' formation that makes us different from growth in size' In general there is little The fifth Processis growth-the increase of tlre form and pattern and the basic during early embryonic development embryoislaiddownonasmallscale,alwayslessthanafewmillimetersinextenl in a variety of ways: cell multiplication' Subsequent$owth can be brought about increaseincellsize,anddepositionofextracellularmaterialssuchasboneand in that differences in growth rates shell. Growth can also m Lorphogenetic body' can generate changes in the overall between organs, or between parts ofthe see.inmore detail in Chapter 12' shapeof the embryo (rig. r'fi), as we shall neither independent of each other nor These five a"rrutop*"L"1 p'ote's"s are however' one can think of pattern formastrictly sequential. In very geileral terms' # tioninearlyd"rrulopm"ntspeci$'ingdifferencesbetweencellsthatleadtochanges But in any real developing system there in form, cell differenti"tor' *i, gro-ttt of events' will be many twists and turns in this sequence gene action 1.e Cell behavior providesthe link between and develoPmental Processes tothe.svnth*it:fp1,"1:-*::Ti":ti|"t;1*:T leads expressionwithincells Gene J;;;;;"'""Jo"i"""r'whichinturnl"t"'**,tl:'::l::it^"::T:'T: ffi "fi ffi;il ffi;ffi nt.t:l"."*-:tv*::*,:?:::':l; pattern ;; ;;; *o""t giventime, which is reflectedin its molecular ft:;il; liifili"i""-;;;;i*ffi any ";roteins*"1'":"ll'-1'-Y-".^:Tt::"-?::3 cellsandtheirprogenyundelgomanychangesinstateasdevelopmentProglesses. othercategoriesofcellbehaviorthatwillconcemusareintercellularcommunicachangesin cell shape and cell movement' tion, also known as cell_cellsignaling, cell proliferation, and cell death' early development are essential for Changing Patterns of gene activity during that determine their future behavior pattern formation. fn"y-gi* cells iientities And' as we saw in the example of and lead evenflrally to thJir nnat differentiation' Fig. f .17 The human embryo changesshape gro*r. Fromthe time the bodyplaniswell "Jia at 8 weeksuntil birth the embryo established panel)' in increases lengthsometenfold(upper to the head whilethe relativeproPortionofthe Asa panel)' (lower restof the bodydecreases Scale changes' embryo ofthe result,the shape 1983' K'L': Moore, = After cm. 10 bar inductionbythesp"**,,'o.ganizer,thecapacityofcellstoinfluenceeachother's By their .",poiaing to signals is crucial for development' fate by producing "rrai" ti"p"' cJh generate the physical forces that bring about movement or change a sheet of cells into a tube' as happens morphogenesis Fig. f .rS)' ftt" t"-"*te -of irl,Xutoltusandothervertebratesduringformationoftheneuraltube(seeBoxlA, generatedby cells changing their shape pp. 4-5), is the result ofcontractile forces atcertainpositionswithinthecelllayer.Animportantfeatureofcellsurfacesis thepresenceofadhesiveproteinsknownascell-adhesionmoleculesthatserve K IT A C ON C E P T U ATOOL L !we rhin t tle ftern rwth I the tPnt. tion, and ales Erall 'nor nTte- ryes here dar mic :Il a dar mic tes. i:aEBt, . for rior eo f cr's kir trrt ETIS IA Tle nis r5e rr a variety of functions: they hold cells together in tissues,they enable cells to sense the nature of the surrounding extracellular rnatrix, and they sewe to guide mrgratory cells such as the neural crest cells ofvertebrates, which leavethe neural tube to form structures elsewhere in the body' Later in development, gfowth involves cell proliferation, which can also influence final form, as parts of the body glow at different rates. The death of cells, known as programmed cell death or apoptosis, is also an intrinsic part of the developmental process; cell death in developing hands and feet helps to form the fingers and toes from a continuous sheet of tissues.We can therefore describe and explain developmental processesin terms of how individual cells and groups of cells behave. Becausethe final structures generated by development are themselvescomposedof cells, explanations and descriptions at the cellular level can provide an account of how these adult structures are formed. Since development can be understood at the cellular level, we can pose the question of how genes control development in a more precise form. We can now ask how genesare controlling cell behavior. The many possible ways in which a cell can behave therefore provide the link between gene activity and the morphology of the adult animal-the final outcome of development. cell biology provides the means by which the genotype becomes translated into the phenotype. r.1o Genescontrol cell behavior by specifyingwhich proteins are made What a cell can do is determined very largely by the proteins plesent within it. The hemoglobin in red blood cells enables them to transPort oxygen; skeletal muscle cells are able to contract because they contain contractile strucfiIres composed of the proteins myosin, actin, and tropomyosin, and othef musclespecific proteins. All these are vely special, or 'luxury" proteins that are not involved in the housekeeping activities that are common to all cells and keep them alive and functioning. Housekeeping activities include the production of energy and the metabolic pathways involved in the breakdown and synthesis of molecules necessaryfor the life of the cell. Although there are qualitative and quantitative variations in housekeepingproteins in different cells, they are not important players in developrnent. In development we are concerned primarily with those luxury or tissue-specificproteins that make cells different from one another. Genes control development mainly by speci8ring which proteins are made in which cells and when. In this sensethey are passiveparticipants in development, compared with the proteins for which they code, which are the agents that directly determine cell behavior, including which genes are exPressed.To produce a particular protein its gene must be switched on and transcribed into messengerRNA (mRNA);the mRNA must then be translated into protein. Both these processes are under severallayers ofcontrol, and translation does not automatically follow transcription. Fig. 1.19 shows the main stagesin gene expression at which the production of a protein can be controlled. For example, nRNA may be degraded before it can be exported from the nucleus. Even if it reachesthe cytoplasm, its translation may be inhibited there. In the eggsof many animals, preformed nRNA is prevented from being ffanslated until after fertilization. Another factor determining which proteins are produced is RNAprocessing.The initial RNAtranscripts of many genes in eukaryotes can be spliced in different ways to give rise to two or mofe diffefent mRNAs; thus a single gene may be able to produce a number of different proteins with different properties. Even if a gene has been transcribed and the mRNA translated, the protein may still not be able to function. Many newly synthesized proteins require further 17 * Fig. 1.18 Localizedcontractionof particular cellscan causea whole sheetof cellsto fold. of a lineof cellsat theirapicesdue Contraction elements to the contractionof cytoskeletal a furrowto formin a sheetof epidermis. causes 18 . l: HtsroRyANDBAstccoNcEprs Fig'r'19 Geneexpressionandproteinsynthesis.Aprotein-codinggenecomprisesastretchof DNAthatcontains a coding region, whichcontains theinstructions formaking theprotein, and adjacent controlregions-promoter andenhancer regions-atwhichthegeneisswitched onoroff. Thepromoter region isthesiteatwhichRNApolymerase bindsandstartstranscribing. Theenhancer regions maybethousands of basepairsdistantfromthepromoter. Transcription ofthegeneintoRNA (1) maybeeither stimulated orinhibited bytranscription factors thatbindto promoter andenhancer regions' TheRNAformedbytranscription isspliced to remove introns(yellow) andprocessed within thenucleus (2)to produce mRNA thatisexported to thecytoplasm (3)fortranslation intoproteinat theribosomes (4).controlof geneexpression andproteinsynthesis occurs mainlyatthelevelof transcription butcanalsooccurat laterstages. Forexample, mRNA maybedegraded beforeit canbe translated' If it isnottranslated immediately it maybestoredin inactive forminthecytoplasm for translation atsomelaterstage. Someproteins require post-translational modification (5)to become biologically active. post-translationalmodification before they acquire biological activity. Reversible post'translational modifications such as phosphorylation can also significantly alter protein function. Alternative RNA splicing and post-translational modification together mean that the number of functionally different proteins that can be produced is considerablygreater than the number ofprotein-coding geneswould indicate-perhaps as much as 10 times more. some genes,such as those for ribosomal RNAs(rRNAs)and transfer RNAs(tRNAs), do not code for proteins; in this casethe RNAsthemserves are the end products. A recently discoveredcategoryof genesis that for microRNAs(miRNAs), small RNA moleculesthat inhibit the translation of specificmRNAs(seechapter 5, Box 58, p.7971. some microRNAs are known to be invorved in gene regulation in development. An intriguing question is how many genes out of the total genome are deveropmental genes-that is, genes specifically required for embryonic deveropment. This is not an easy estimate to make. In a few particularly well studied caseswe have rough estimates of the minimum number of genes involved in a particular aspectof development.In the early developmentof Drosophilaat least 60 genesare directly involved in pattern formation up to the time that the embryo becomes divided into segments.rn caenorhabditis, at least 50 genesare neededto speci$ra small reproductive structure known as the vulva. All these are quite small numbers comparedto the thousandsof genesthat are active at the ,.*" ti-"; someof these are essentialto development in that they are necessary for maintaining life, but provide no or little information that influences the course of development. Recent studies have shown that many genes change their activity during deveropment, and as the total number of genesin the nematode and Drosophilais around 19,000 and 20'000, respecfively,the total number ofgenes involved in development is many thousands.A systematicanalysis of nearly 9o%of the nematod"', g"r", showed that 7722 genes,at least, were involved in deveiopment. Developmentar genes typically code for proteins involved in the regulation of cell behavior-receptors, growth factors,intraceilular signalingproteins, and generegulatory proteins. Many ofthese genes,especiallythose for receptorsand signaling molecules,are used throughout an organism's life, but others are active only during embryonic development. r.tt The expressionof developmentalgenesis underthe control of complex control regions All the somatic cells in an embryo are derived from the fertilized egg by successive rounds of mitotic cell division. Thus, with rare exceptions, they all contain identical TooL Krr A coNcEPTUAL r dtrf 'RNA lrc€f !t'n nat nbe r IIE ible ntly tion nbe urld IAs), ECtS. TNA 1e7l,. . lopENL I \A/e dar I are mes ify a bers ke but rFnt Ent, Ino ]tny red nof !ne. palmly ,Eeneticinformation, the same as that in the zygote. The differences between cells must therefore be generatedby differences in gene activity that lead to the synthesis ofdifferent proteins. Turning the correct geneson or offin the colTectcells at the €orrect time becomesthe central issue in development.The genesdo not provide a blueprint for development but a set of instructions. Key elements in regulating tie readout of these instructions are the control regions located adjacent to most tuxury genes and developmental genes.These are acted on by gene-regulatory proteins, or transcription factors, which switch genes on or off by, respectively, activating or repressing transcription. Some gene-regulatory proteins act by hinding directly to the control regions (seeFig. 1.19)whereas others interact with tmnscription factors already bound to the DNA. Developmental genes are highly regulated to ensure they are switched on only at the right time and place in development. This is a fundamental feature of development. To achieve this, they usually have extensive and complex control regions composedof one or more regulatory modules, known as cis-regulatorymodules tthe 'cis'refers to the fact that the regulatory module is on the sameDNA molecule as the gene it controls). Each module contains multiple binding sites for different ranscription factors, and the combination of factors that binds determines whether the gene is on or off. On average,a module will have binding sites for four to eight different transcription factors. Different genes can have the same control module, which usually means that tbey will be expressedtogether, or different genesmay have modules that contain some, but not all, of the binding sites in cortmon, introducing subtle differences in the timing or location of expression.A gene with more than one regulatory module can be expressedat different times and placesduring development, depending on which module predominatesat any given time in directing gene expression. Thus, an organism's genes are linked in complex interdependent networks of expressionthrough their regulatory modules and the proteins that bind to them. Common examples of such regulation are positive feedback and negative feedbackloops in which a transcription factor respectivelypromotes or represses the expressionof a genewhose product maintains that gene expression(Fig.1.20). Lrdeed,the network ofinteractions between gene-regulatorymodules that has been describedfor early sea urchin development is quite bewildering at first, or even second,sight, as we shall seein Chapter 5. As all the key steps in development reflect changes in gene activity, one might be tempted to think of development simply in terms of mechanismsfor controlling gene expression.But this would be highly misleading. For gene expressionis only the first step in a cascadeof cellular Processesthat lead via protein synthesis to changesin cell behavior and so direct the course of embryonic development. To think only in terms of genesis to ignore crucial asPectsof cell biology, such as change in cell shape, that may be initiated at several steps removed from gene activity. In fact, there are very few caseswhere the complete sequenceof eventsfrom gene expression to altered cell behavior has been worked out. The route leading from gene activity to a structure such as the five-fingered hand may be tortuous. t.rz Developmentis progressiveand the fate of cellsbecomesdetermined at different times dlve tl:al II As embryonic development proceeds,the organizational complexity of the embryo becomes vastly increased over that of the fertilized egg. Many different cell types are formed, spatial patterns emerge, and there are major changesin shape.All this occurs more or less gradually, depending on the particular organism. But, in 19 Postitivefeedback adivator I lr'J r-' w.@. gene2 gene l adivator I lrlff" WjW gene4 gene3 W tr Fig.1.2O Simplegeneticfeedbackloops. Top:gene'l isturnedon byanactivating factor(green); the proteinit transcription gene2. TheProtein produces (red)activates productof gene2 (blue)not onlyactson gene1, furthertargets,but alsoactivates feedback loopthatwill keep forminga positive genes1 and2 switched on evenin the absence Bottom:whenthe of the originalactivator. productof the geneat the endof the pathway feedback inhibitsthe firstgene,a negative loopisformed.Arrowsindicateactivation; inhibition. barredlineindicates 20 . r: HrsroRYANDBAstccoNcEPTs Fig. 1.21 Thedistinction betweencell fate, determination,and specification.In this idealizedsystem,regionsA andBdifferentiate into two differentsortsof cells,depictedas Thefatemap(firstpanel) andsquares. hexagons lf cells showshowtheywouldnormallydevelop. from regionBaregraftedinto regionA andnow developasA-typecells,the fate of regionBhas (secondpanel).By notyet beendetermined determined if regionBcellsarealready contrast, whentheyaregraftedto regionA, theywill developasBcells(thirdpanel).Evenif Bcellsare in that theymaybespecified, not determined, theywillform Bcellswhenculturedin isolation fromthe restof the embryo(fourthpanel). general, the embryo is first divided up into a few broad regions, such as the future germ layers (mesoderm,ectoderm, and endoderm).Subsequently,the cells within these regions have their fates more and more finely determined' Mesoderm, for example, becomesdifferentiated into muscle cells, cartilage cells, bone cells, the fibroblasts of connective tissue, and the cells of the dermis of the skin. Determination implies a stable changein the internal state of a cell, and an alteration in the pattern of gene activity is assumed to be the initial step, leading to a change in the proteins produced in the cell. It is important to understand clearly the distinction between the normal fate of a cell at any parLicular stage,and its state of determination. The fate of a group of cells merely describes what they will normally develop into. By marking cells of the early embryo one can find out, for example, which ectodermal cells will normally give rise to the nervous system, and of those, which to the retina in parLicular. However, that in no way implies that those cells can only develop into a retina, or are already committed, or determined, to do so. A group ofcells is called specified if, when isolated and cultured in the neutral environment of a simple culture medium away from the embryo, they develop more or less according to their normal fate (Fig. 1.21).For example, cells at the animal pole of the amphibian blastula (seeBox 1'{, pp. 4-5) are specifiedto form ectoderm, and will form epidermis when isolated. Cells that are specified in this technical senseneed not yet be determined, for influences from other cells can change their normal fate; if tissue from the animal pole is put in contact with cells from the vegetalpole, the ani.malpole tissuewill form mesoderminsteadof epidermis. At a later stage of development, however, the cells in tJre animal region have become determined as ectoderm and their fate cannot then be altered. Tests for specification rely on culturing the tissue in a neutral environment lacking any inducing signals,and this is often difficult to achieve. The state of determination of cells at any developmental stage can be demonstrated by transplantation experiments. At the blastula stage of the amphibian K tT L A C ON C E P TUATOOL prcsumPwe eyeregron presqmptivs eprderm6 0resumDllve 'endoilerm presumplve plate neural tf- I Iailbud-stageembrYo presumptive \mesoderm blaslopore fitg.1.22 Determinationof the eye region with time in amphibiandevelopment.lf the thatwill normallygiverise regionof the gastrula to aneyeisgraftedintothetrunkregionof a neurula(middlepanel),the graftforms suchas typicalof its newlocation, structures notochordandsomites.lf, however'the eye regionfrom a neurulaisgraftedintothe same asaneyeJike site(bottompanel),it develops it has stage later at this since structure, becomedetermined. bre rhin ErIn plls, *infterto a frte |Pof rells rwill E io rinto ffiaI rlop t the brm r this D CAII rcells edderrhalr ts for g eny lEloIF frian ri 21 side of embryo, one can graft the ectodermal cells that give rise to the eye into the is, that new their to Position; de body and show that the cells develop according At this (Fig. 7.22l|. somites into rnesodermalcells like those of the notochord and early stage,their potential for development is much greater than their normal fate' Itrowever,if the same operation is done at a later stage,then the future eye region yill form structules tyPical of an eye. At the earlier stage the cells were not yet determined as eye cells, whereaslater they had become so' It is a general feature of development that cells in the early embryo are less more and uarrowly determined than those at later stages;with time, cells become that determination assume We potential. developmental more restricted in their fovolves a change in the genes that are expressedby the cell and that this change rmxes or restricts the cell's fate, thus reducing its developmentaloptions. We have already seen how, even at the two-cell stage,the cells of the seaurchin rmbryo do not seem to be determined. Each has the potential to genefate a whole much uE\vlarva (seeSection1.3).Embryoslike these,where the potential of cells is Vertebrate regulative' termed are fate, &Featerthan that indicated by their normal cmbryos are among those capable of considerable regulation. In contrast, those to rmbryos where, from a very early stage, the cells can develop only according has mosaic the term 1.3, rfreir early fate are termed mosaic,As we saw in Section rLbng history. It describes eggs and embryos that develop as if their pattern of ffiuturedevelopment is laid down very early, even in the egg, as a mosaic of differrnt molecules. The different parts of the embryo then develop quite independently demardeach other. In such embryos, cell interactions may be quite limited. Ttre oafion between the regulative and mosaic strategies of development is not always frarp, and in part reflects the time when determination occurs; it occurs much emlier in mosaic systems. 22 . r: HrsroRYANDBAstccoNcEPTs The difference between regulative and mosaic embryos reflects the relative importance of cell-cell interactions in each system. The occurrence of regulation absolutely requires interactions between cells, for how else could normal develop ment take place and deficiencies be recognized and restored? A truly mosaic embryo, however, would in principle not require such interactions. No purely mosaic embryos are known to exist. 1.r3 Inductiveinteractionscan make cellsdifferent from eachother Fi9.1.23 An inducingsignalcan be transmitted from one cell to anotherin three mainways.Thesignalcanbea diffusible witha receptor on the molecule. whichinteracts (secondpanel),or the signal targetcellsurface canbe producedby directcontactbetween proteinsat the cell two complementary (thirdpanel).lfthe signalinvolves surfaces it maypassdirectlyfrom cell smallmolecules to cellthroughgapjunctionsin the plasma (fourthpanel). membrane Making cells different from one another is cenffal to development. There are numerous examples in development where a signal from one group of cells influencesthe development of an adjacent group of cells. This is known as induction and the classic example is the action of the Spemann organizer in arnphibiens (see Section 1.4). Inducing signals may be propagated over several or even many cells, or be highly localized. The inducing signals from the amphibian organizer affect many cells, whereas other inducing signals may passfrom a single cell to its immediate neighbor. T\uo different types of inductions should be distinguished: permissiveand instructive. Permissiveinductions occur when a cell makes only one kind of response to a signal, and makes it when a given level of signal is reached. By contrast, in an instructive induction, the cells respond differently to different concenffations of the signal. There are three main ways in which inducing signals may be passedbetween cells (Fig. 1.23).First, the signal can be transmitted through the extracellular space, usuallybymeans of a secreteddiffirsible molecule. Second,cells mayinteract directly with each other by means of molecules located on their surfaces. In both these cases,the signal is generally receivedby receptor proteins in the cell membrane and is subsequently relayed through intracellular signaling systems to produce the cellular response.Third, the signal may passfrom cell to cell directly. In animal cells, this is through gap junctions, which iue specializedprotein pores in tJre apposedplasma membranes providing direct channels of communication between the rytoplasms of adjacent cells through which small molecules can pass. Plant cells are connected by strands of cytoplasm called plasmodesmata,through which even quite large molecules such as proteins can passdirectly from cell to cell. In the caseof signaling by a diffirsible molecule or by direct contact, the signal is received at the cell membrane. If it is to alter gene expression in the nucleus, the signal has to be transmitted from the membrane to the cell's interior. This process is known generally as signal transduction, and is carried out by relays of intracellular signaling molecules that are activated when the extracellular signal molecule binds to its receptor. Intracellular signaling proteins and small-molecule second messengerssuch as cyclic AMP interact with one another to transmit ti.e signal onward in the cell. Activation and deactivation of the pathway components by phosphorylation is an important feature of most signaling pathways. In the case of development, most of the signaling we are concerned with leads to gene activation or repression, but signaling pathways are also used to temporarily alter enzyme activity and metabolic activity within cells and to relay nerve impulses. The variety of signals a cell may be receiving at any one time are integmted by cross-talk between the different signaling pathways to produce an appropriate response. An important feature of induction is whether or not the responding cell is competent to respond to the inducing sigrral.This competencemay depend, for example,on the presenceof the appropriate receptor and transducing mechanism, or on the presenceofthe particular transcription factors neededfor gene activatiorl A cell's competence for a particular response can change with time; for example, K IT A C ON C E P TUATOOI L htive trion rclop psaic urely t are influrtion, bians many rnizer I to its bhed: s only pal is ltly to tween sPace, irectly , these brane mduce rnimal in the ltween - Plant which t: signal ns, the lrOCeSS h:n of : signal olecule nit the Etrents he case :activanzyme Yadety rs-talk te. ; cell is nd, for nnism, iration. emple, IT rhe Spemann organizer can induce changes in the cells it affects only during a restricted time window [n embryos, it seemsthat small is generally beautiful where signaling and Pattem formation are concefned. Whenever a Pattern is being specified, the size of the group of cells involved is barely, if ever, greatel than 0.5 mm in any direction: that is, some 50 cell diameters. Many patterns ale specified on a much smaller scaleand hvolve just tens or a few hundreds of cells. This means that the inducing signals involved in pattem formation reach over distances of the order of only 10 times a cell diameter. The final olganism may be very big, but this is almost entirely due to growth of the basic Pattern. t.t4 The responseto inductive signalsdependson the state of the cell Inductive signals can alter how the induced cells develop. They can thus be regarded as providing the cells with instnrctions as how to behave.After receiving an inductive signal, a cell usually then develops autonomously that is' without new signals from another cell, for some time' It is important to realize that the Fesponseto inductive signals is entirely dependent on the current state ofthe cell. llot only does it have to be competent to respond, but the number of possible responsesis usually very limited. An inductive signal can only select one response from a small number of possible cellular responses. Thus instructive inducing signals would be more accurately called selective signals. A truly instructive signal would be one that provided the cell with entirely new information and capabilities' by providing it with, for example, new genes,which is not thought to occtll during development. The fact that, at any given time, an inductive signal selects one out of several possible responseshas several imPortant implications for biological economy' On ttre one hand, it means that different signals can activate a particular gene at different stages of development: the same gene is often turned on and off repeatedly during development. On the other, the same signal can be used to elicit different responsesin different cells. A particular signaling molecule' for example, can act on severaltypes ofcell, evoking a characteristicand different responsefrom each, depending on their developmental history. This enables the fixed set of genes and signals to be related to each other in a variety of different combinations' effectively expanding the number of developmentally distinct signals and responsesthat can be made by cells. As we will see in future chapters, evolution has been lazy with respect to this aspect of development, and the same basic intracellular signaling pathways are used again and again for different purposes' 1.rs Patterningcan involve the interpretation of positional information One general mode of pattem formation can be illustrated by considering the patterning of a simple non-biological model-the French flag (Fig. 7.24).T\e French flag has a simple pattem: one-third blue, one-third white, and one-third red, along just one axis. Moreover, the flag comes in many sizesbut always the same Pattern' and thus can be thought of as mimicking the capacity of an embryo to regulate. Given a line of cells, any one of which can be blue, white, or red, and given also that the line of cells can be of variable length, what sort of mechanism is required for the line to develop the pattern ofa French flag? One solution is for the group of cells to acquire positional information' That is' each cell acquires an identity, or positional value, that is related to its position along the line with respect to the boundaries at either end' After they have Fig.1.24 TheFrenchflag. 23 24 I: HIS T O RY AND BASIC CONCEPTS acquired their positional values, the cells interpret this information by differentiating according to their genetic program. Those in the left-hand third of the line will become blue, those in the middle third white, and so on. Pattern formation using positional information thus implies at least two distinct stages:first the positional value has to be specified with respect to some boundary, and then it has to be interpreted. The separation of these two Processeshas an important implication: it means that there does not need to be a set relation between the positional values and how they are interpreted. In different circumstances,the same set of positional values could be used to generate the Italian flag or another pattern. How positional values will be interpreted will depend on the particular genetic instructions active in the group of cells and will be influenced by their developmental history. Cells could have their position specified by various mechanisms. The simplest is based on a gradient of some substance. If the concentration of some chemical decreasesfrom one end of a line of cells to the other, then the concentration of that chemical in any cell along the line effectively specifies the position of the cell with respect to the boundary (Fig. 7.25). A chemical whose concentration varies and which is involved in pattern formation is called a morPhogen.In the caseof the French flag, we assume a source of morphogen at one end and a sink at the other, and that the concentrations ofmorphogen at both ends are kept constant but are different from each other. Then, as the morphogen diftrses down the line, its concentration at any point effectively provides positional information. If the cells can respond to threshold concentrations of the morphogen-for example, above a particular concentration the cells develop as blue, while below this concentration they become white, and at yet another, lower, concentration they become red-the line of cells will develop as a French flag (seeFig. 1.25).Thresholds can represent the amount of morphogen that must bind to receptors to activate an intracellular signaling system, or concentrations of transcription factors required to activate particular genes. The use of threshold concentrations of transcription factors to speciSrposition is most beautifully illustrated in the early development of Drosophila,as we shall see in Chapter 2. Other ways of speciffing positional information are by direct intercellular interactions and by timing mechanisms, as discussedin Chapter 4 and elsewherein the book. The French flag model illusffates two important features of development in the real world. The first is that, even if the lengttr of the line varies, the system regulates and the pattern will still form correctly, given that the boundaries ofthe system are properly defined by keeping the different concentrations ofmorphogen constant at either end. The second is that the system could also regenerate the complete original pattern if it were cut in half, provided tfiat the boundary concentrations were re-established. It is therefore truly regglative. We have discussedit Fig. 1.25 The Frenchflag model of pattern formation. Eachcellin a lineof cellshasthe potential gradientof some to a concentration to developasblue,white,or red.Thelineof cellsisexposed positional concentration at that point.Each value defined by the acquires a substance andeachcell intoblue,white,or red, anddifferentiates valueit hasacquired celltheninterprets the positional that flagpattern.Substances geneticprogram, thusformingthe French according to a predetermined of Thebasicrequirements of cellsin thiswayareknownasmorphogens. candirectthe development at eitherendof the gradientmustremain of substance sucha systemarethat the concentration to the system.Eachcellmustalso differentfrom eachotherbut constant,thusfixingboundaries containthe necessary informationto interpretthe positionalvalues.Interpretationofthe positional of morphogen. to differentconcentrations valueis basedupondifferentthresholdresponses K IT A C ON C E P TU ATOOL L trF lne TKT ay" ia n tbn IIIF 8ac the ilb"v 5t is d{-el no f : cell ries ro f t tie lant line, f the Eple, tctnilrme I €an !e an uircd ption ment tioDal isms, nt in Ftem of tle hogen F the flcen$ed it E ttial E tacft d. Ef that rnts of il bo lbnal IT 25 here as a one-dimensionalpatterning problem, but the model can easily be extended to provide two-dimensionalpatterning (Fig.1.26). br many casesit is still undear how positional information is specified-molphogen diffirsion is but one of tJremechanisms tlrat have been discovered-but the evidence for it is strong. However, we do not lrrow how fine-grained the differences in position are. For example, has every cell in the early embryo a distinct positional value of its own and is it able to make use of this difference? An important aspect of many patterning processesis the setting up of a polarity in the plane of a sheet of cells. It is as if a direction arrow is Presentin all the cells in the sheet making one end slightly different from the other, rather like a compass needle pointing north. This type of polarity is known as planar cell polarity and is discussed further in Chapters 2 and 7. One obvious example of planar cell polarity is the hairs on the Drosopltilawing, which all point in the same direction. r.r6 Lateralinhibition can generatespacingPatterns Many structures,such as the featherson a bird's skin, afe found to be more or less regularly spaced with respect to one another. One mechanism that gives rise to such spacing is called lateral inhibition (Fig. 1.27).Given a gloup of cells that all have the potential to differentiate in a particular way, for example as feathers, it is possible to regularly space the cells that will form feathers by a mechanism in which cells that.begin to form feathers inhibit the adjacent cells from doing so. This is reminiscent of the spacingof trees in a forest being causedby competition for sunlight and nutrients. In embryos, lateral inhibition is often the result of the differentiating cell secreting an inhibitory molecule that acts locally on the nearest cell neighbors to prevent them developing in a similar way. Flg. 1.25 Positionalinformation can be used to generatePatterns.A goodexample,as shownhere,iswherethe peopleseatedin a stadiumeachhavea positiondefinedbytheir Eachpositionhasan rowandseatnumbers. aboutwhichcoloredcardto holdup, instruction andthismakesthe pattern.lf the instructions werechanged,a differentpatternwouldbe canbe information formed,andsopositional usedto produceanenormousvarietyof patterns. !.17 Localizationof cytoplasmicdeterminantsand asymmetriccell division can make cells different from each other Positional specification is just one way in which cells can be given a particular identity. A separatemechanism is basedon cytoPlasmiclocalizationand asymmetric cell division (Fig.1.28).Asymmetric divisions are so called becausethey result in daughter cells having properties different from each other, independently ofany environmental influence. The properties of such cells therefore depend on their lineageor line of descent,and not on environmental cues.Although some aspnmetric cell divisions are also unequal divisions in that they produce cells of different sizes, this is not usually the most important feature in animals; it is the unequal distribution of cytoplasmic factors that makes the division asymmetric. An alternative way of making the French flag pattern from the egg would be to havechemical differences(representingblue, white, and red) distributed in the egg fo the form of determinants that foreshadowed the French flag. When the egg underwent cleavage, these cytoplasmic determinants would become distributed :moilg the cells in a particular way and a French flag would develop. This would require no interactions between the cells, which would have their fates determined fum the beginning. Although such extreme examplesof mosaic development are not known in nature, there are well known caseswhere eggs or cells divide so that some cytoplasmic determinant becomes unequally distributed between the two daughter cells and frey develop differently. This happens at the first cleavage of the nematode egg, ior exarnple, and defines the antero-Posterior axis of the embryo. The germ cells of Fig.1.27 Lateralinhibition can give a spacing pattern.Lateral inhibitionoccurswhen producean inhibitorthat structures developing prevents the formationof anysimilarstructures to them,andthe structures in the areaadjacent becomeevenlyspacedasa result. 26 I: H IS T OR Y AND EASIC CONCEPTS Drosoythilaare also specified by cytoplasmic determinants, in this casecontained in the cytoplasm located at the posterior end ofthe egg, and in Xenoptsakey deter_ minant in the first stagesof embryonic development is a protein t*o* as vegT, which is localized in the vegetal region of the fertilizea ejg. In generar, however, as developmentproceeds,daughter cellsbecomedifferent from eachother because of signals from other celrs or from their extracellular environment, rather than becauseof the unequal distribution of cytoplasmic determinants. A very particular qpe of asymmetric division is that of stem cells, which are cells that are capable of repeated division and that produce daughter cens, one of which remains a stem ceil while the other differentiates into a iariety of cell types (Fig.1.29).stem cells that are capableofgiving rise to all the cell types in the body are present in embryos, while in adults, stem cells of apparently more rimited potential are responsiblefor the continual renewal of tissuessuch asblood, epidermis, and gut epithelium and for the replacement of tissues such as muscle when required. The difference in the daughter cells can be due either to asyrnmetric distribution of cytoplasmic determinants or to the effects of external signals. The generation of neurons in Drosophila,for exampre, depends on the distribution of cytoprasmic determinants in neuronal stem cells. Stem cells that can give rise to all other types of cells,but not to a complete embryo, are known as pluripotent. The fertilized egg, which can give rise to a complete embryo, is described as totipotent. t 'ra The embryo contains a generative rather than a descriptive Flg. 1.28 Cell division with asymmetric of cytoplasmic determinants, lf a partiotlar molecule is distributed unevenly in distribution the parent cell, cell dMsion will result in its being shared unequally betrrveenthe cytoplasm ofthe two daughter cells. The more localizedthe cytoplasmic determinant is in the parental cell, the more likelyit will be that one daughtercell will receiveall of it and the other none,thus producing a distinct difference between them. program All the information for embryonic development is contained within the fertilized egg' so how is this information interpreted to give rise to an embryo?one possibility is that the structure ofthe organism is somehow encoded as a descriptive program in the genome. Does the DNA contain a furl description of the organism to which it will give rise: is it a blueprint for the organism? The answer is no. Instead, the genome contains a program of instructions for making the organism_a generative program-in which the cytoplasmic constituents of eggs and cells are essential players along with the genes. A descriptive program such as a blueprint or a plan describes an object in some detail, whereas a generative program describes how to make an oulect. For the same object the programs afe very different. consider origami, the art of paper folding. By folding a piece of paper in various directions it is quite easy to make a paper hat or a bird from a single sheet. To describe in any detail the finar form of the paper with the complex relationships between its parts is really very difficult, and not of rruch help in explaining how to achieve it. Much rnore useful and easier to forrnulate are instmctions on how to fold the paper. The reason for this is that simple instructions about folding have complex spatial consequences.In deverop ment' gene action similarly sets in motion a sequence of events that can bring about profound changesin the embryo. one can thus think of the genetic information in the fertilized egg as equivalent to the folding instructilns in origami; both contain a generative program for making a particular structure. A further distinction can be made between the embryo,s genetic program and the developmental program of a particular cell or group of ce[s. The genetic program refers to the totality of information provided by the genes, whereas a developmental program may refer only to that part of the genetic program that is controlling a particular group of ce[s. As the embryo develops, diFerent parts acquire their own developmental programs as a result of cell-cell interactions and the activities of selectedsets of genes.Each celr in the embryo thus has its own developmental program, which may change as development proceeds. K IT A C ON C E P TU ATOOL L t-r p ttE gn" dk :d p(5 odr lrFd Eis, iedtbn no f IEic t?es .€' ilized bility Eram rhich rL the rative rntial lsome irr the 'paper r make irm of iltrcult, I easier i is tfiat hreloP n bring nformarigami; am and genetic hereas a n that is nt parts ions and l its own il t.tg The reliability of development is achievedby a variety of means The development of embryos is remarkably consistent and reliable; you need only look at the similar lengths of your legs, each of which developsquite independently of the other for some 15 years (Chapter 9). Embryonic development needsto be reliable in the sensethat the adult organism can function properly. For example, both wings of a bird must be very similar in size and shape if it is to be able to fly satisfactorily. How such reliability is achieved is an important problem in development' Developrnent needs to be reliable in the face of fluctuations that occur either within the embryo or in the external environment. Internal fluctuations include small changesin the concentrations of molecules,and also changesdue, for example, to mutations in genes not dfuectly linked to the development of the organ in question. External factors that could perturb development include temPerature and environmental chemicals. One of the central problems of reliability relatesto pattern formation. How are cells specifiedtobehaveinaparticularwayina partiorlarposition?TWowaysinwhichreliability is assured ale aPparent redundancy of mechanism and negative feedback' Redundancyis when there are two or more ways of carrying out a particular process; if one fails for any reason another will still function. It is like having two batteries in your car instead of just one. True redundancy-such as having two identical genesin a haploid genome with identical functions-is IaIe except for casessuch as rRNA' where there can be hundreds of similar genes.Apparent redundancy, on the other hand, where a given Processcan be specifiedby severaldifferent mechanisms,is probably one of the ways that embryos can achieve such precise and reliable results. It is like being abte to draw a straight line with either a ruler oI a piece of taut string. This type of situation is not true redundancy, but may give the impression of being so if one mechanism is removed and the outcome is still apparently normal' Negative feedback also has a role in ensuring consistency; here, the end product ofa processinhibits an earlier stageand thus keepsthe level ofproduct constant (see,for example, Fig. 1.20).The classicexample is in metabolism, where the end product of a biochemical pathway inhibits one of the enzymes that act early in the pathway. Yet another reliability mechanism relates to the complexity of the networks ofgene activity that operate in development. There is evidence that these networks are robust and relatively insensitive to small changes in, for example, the rates of the individual processesinvolved. 1.20 The complexity of embryonic development is due to the complexity of cells themselves Cells are, in a way, more complex than the embryo itself. In other words, the network of interactions between proteins and DNA within any individual cell contains many more components and is very much more complex than the interactions between ttre cells of the developing embryo. However clever you think cells are, they are almost always far cleverer. Each of the basic cell activities involved in development,such as cell division, fesPonseto cell signals,and cell movements, are the result of interactions within a population of many different intracellular proteins whose composition varies over time and between different locations in the cell. Cell division for example, is a complex cell-biological Program that takes Place over a period of time, in a set order of stages,and requires the construction and preciseorganization of specializedintracellular structures at mitosis. Any given cell is expressing thousands of different genes at any given time. Much of this gene expression may reflect an intrinsic pfoglam of activity' 27 Stemrells a'@ -oo b "(_ oi 'o io Fig.1.29 Stemcells.Stemcells(S)arecells andgiveriseto that both renewthemselves celltypes.Thus,a daughtercell differentiated arisingfromstemcelldivisionmayeither developinto anotherstemcellor giveriseto a quitedifferenttype of cell(X).lt mayalsodivide to giveriseto two stemcellsor to two different typesof cells(XandY). 28 . r: HtsroRYANDBAslcc oNc EPTS independent of external signals. It is this complexity that determines how cells respond to the signals they receive; how a cell lesponds to a Particular signal depends on its internal state. This state can reflect the cell's developmental history-cells have good memories-and so different cells can respond to the same signal in very different ways. We shall see many examples of the same signals being used over and over again by different cells at different stages of embryonic development, with different biological outcomes. We have at present only a fragmentary picture of how all the genes and proteins in a cell, let alone a developing embryo, interact with one another. But new technologies are now making it possible to detect the simultaneous activity of hundreds of genes in a given tissue. The discipline of systems biology is also beginning to develop techniques to reconstruct the highly complex signaling networks used by cells. How to interpret this information, and make biological sense of the patterns of gene activity revealed, is a massive task for the future. !l il (4Ir I l rfltlfllilm riLI'nl|],tilft 1 l u , L rt g ;iilrlli:lglllllT l1l-tudfl4tt lll ""lltr' ll ru,tTlN -rl|lliilllm t'( tliill --rmfln q iilul'l 1Lllflmli iiiii|llrfiu0 ur{c,i 1 Surnrnary iriNlllirlll .tr of cells.ThemajorProcesses behavior resultsfromthe coordinated Development iill|" m0 or are cell division,Patternformation,morphogenesis involvedin development groMh. Genes cell migration,cell death,and changein form, celldifferentiation, andthus aresynthesized, controlcellbehaviorbycontrollingwhereandwhenProteins processes. cell biologyprovidesthe link betweengene action and developmental in theirshape, in the genestheyexpress, Duringdevelopment, cellsundergochanges and in their in the signalsthey produceand resPondto, in their rateof proliferation, arecontrolledlargelyby the presof cellbehavior Alltheseaspects migratorybehavior. enceof specificproteins;geneactivitycontrolswhich proteinsare made.Sincethe somaticcellsin the embryogenerallycontainthe same geneticinformation,the activityof selected arecontrolledbythe differential that occurin development changes to setsof genesin differentgroupsof cells.Thegenes'controlregionsarefundamental at determined becomes fate of cells progressive the process. and is Development this is embryo usually in the early cells of potential development The for differenttimes. much greaterthan their normalfate,but this potentialbecomesmore restrictedas involvingsignalsfromonetissueor cell proceeds. Inductiveinteractions, development to another,areoneof the mainwaysof changingcellfateanddirectingdevelopment. distributed areunequally comPonents celldivisions, in whichcytoplasmic Asymmetric means of pattern to daughtercells,can also makecellsdifferent.One widespread positional valuewith cellsfirstacquirea information; generation isthroughpositional valuesby behavingin differrespectto boundaries andthen interprettheirpositional choosingoneor thaninstructive, signalsaremoreselective ent ways.Developmental other of the developmental Pathwaysopen to the cell at that time. The embryo for program-it is morelikethe instructions not a descriptive, containsa generative, includA varietyof mechanisms, by paperfoldingthana blueprint. makinga structure areinvolvedin makingdevelopment andnegativefeedback, ingapparentredundancy lieswithinthecells. of development remarkably reliable. Thecomplexity All the informationfor embryonicdevelopmentis containedwithin the fertilized egg-the diploidzygote.The genomeof the zygotecontainsa programof instrucprogram, tionsfor makingthe organism.In the workingout of this developmental of the egg and of the cellsit givesriseto areessential the cytoplasmicconstituents i r" ll1ll .i ( " lilLL ifl lllll . illllll FU R T H ERRE A D IN G EIIS Fal @1 tne rioC mic Eins ts\f, tr of EIF Ets trhe I 3 F yr F Ps F FF playersalong with the genes.Strictly regulatedgene activity, by controllingwhich proteinsare synthesized,and where and when, directs a sequenceof cellular activitiesthat bringsabout the profoundchangesthat occurin the embryoduring development.The major processesinvolvedin developmentare cell division, cellmigration,celldeath,and celldifferentiation, patternformation,morphogenesis, growth.All of theseprocesses canbe influencedby communicationbetweenthe cells of the embryo. Developmentis progressive,with the fate of cellsbecominglrnre Cellsin the earlyembryousuallyhave preciselyspecifiedasdwelopmentproceeds. potential for developmentthan is evident from their normal fate, a much greater and this enablesembryosto developnormallyevenif cellsare remwed, added,or potentialbecomesmuch transplantedto differentpositions.Cells'developmental proceeds. Manygenesareinvolvedin controllingthe morerestrictedasdevelopment complexinteractionsthat occurduringdevelopment,and reliabilityis achievedin ofanimalsandplants, ofgenescontrolthe development a varietyofways.Thousands and a full understandingof the processesinvolvedis far from beingachieved.While basicprinciplesand certaindevelopmentalsystemsare quite well understood,there arestillmanygaps. FURTHER READING The origins of developmental biology F F" Cole, F.l.: Eorly Theoriesof SexuolGmration. Oxford: Clarendon Press. 1930. Hamburger, Y.: TheHerttuge of Experimmtal Embryolagt: Hans Spemannondthe OrganiTrr. NewYork: Oxford University Press' fb 1988. Milestones in D 6relof,ment F- ts !lv la tr FtFd Fin Fft F F{r rp lbt l+ F* I t I F. t* Irr. tIral i II [http:/lriuww.nature.corn/milestones/development/index.htrn! Needham, J.: A Histo'ry of Enfuryolog. Cambridge: Cambridge University Press, 1959. effects" in and "assimilating Sander, K.: "Mosaic worlf after embryogenesis: Wilhelm Roux's condusions Arch. D4,. Biol. 1997, disabling frog blastometes. Rotu<'s 2OO:237-239. Sander, K.: Shaking a oonaept flens f)riesch and the varied fates of sea uff:hin blastomeres. Rotn's Arch. Dett Biol. 7992' z()li 265-267. Wilson, E.B.: TheCel in Developmentond Heredity. New York: Macmillan. 1896. Wolpert, L.: Evolution of tlre cell theory. PhiI. Trors. Roy.Soc. Lond.B 1995, 349t 227-233. A conceptual tool kit Alberts, 8., et aL: EssentialCeIIBiologt: An Introduchon to the Moleaiar Biologr of the Ce'il.2nd edn. New York: Garland Science. 2003. splicing: increasing diversity Graveley, B.R: Alt€mative TrendsC'enet.2007, a7: 7oo-1o7. in the proteornic wotlil Howard, M.L., Davidson, E.H.: cis-Regulatory aontrol circtrits in developrnent. Dst. Biol. 2oM,271: 109-118. Jordan, J.D., Landau, E.M., Lyengar, R.: Signaling networks: CeI] 2ooo, lO3: the origins of ce[ular multitasking. 193-200. and animal rcgulation Levine, M., Tjian, R: ltanscriptional diversity. Nature 2OO3,424: 747 -157. of corre mechanisms to generate Nelson, WJ.: Adaptation polarity. Notttre 2oo3, 422: 766-77 4. Papin, JA., Hunter, T., Palsson, B.O., Subramanian, S.: networks and of cellular sip.alling Reconstnrction analysis of their properties. Nat Rev.MoI. Biol. 2oo5, 6l 99-777. development? wolpert, L.: Do we understand 266i 571-572. Wolpert, L.: One hundred years of positional TrendsGenet.1996. a2: 359-364. Science1994' information. 29