The as a Model Organism

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					FIRSTHAND KNOWLEDGE                                                                                                                                                                                                                                                    DEVELOPMENTAL BIOLOGY

                                                                                                                                                                              The                                                                       el organism that would allow him to
                                                                                                                                                                                                                                                        easily study the genetic control of de-

                                                                                                                                                                                                                                                        velopment in vertebrates, including
                                                                                                                                                                                                                                                        humans. As an aquarium enthusiast,
                                                                                                                                                                                                                                                        he soon hit upon the zebrafish, which
                                                                                                                                                                                                                                                        combines two important characteris-

                                                                                                                                                                              as a Model
                                                                                                                                                                                                                                                        tics: it is relatively easy to use in ex-
                                                                                                                                                                                                                                                        periments, and as a vertebrate, its de-
                                                                                                                                                                                                                                                        velopment is very similar to that of
                                                                                                                                                                                                                                                        humans – far more so, for instance,
                                                                                                                                                                                                                                                        than that of fruit flies, another im-

                                                                                                                                                                              Organism                                                                  portant model organism.
                                                                                                                                                                                                                                                           Despite obvious differences, ze-
                                                                                                                                                                                                                                                        brafish have a lot in common with
                                                                                                                                                                                                                                                        us. Most of the organs and cell types
                                                                                                                                                                                                                                                        that occur in humans are likewise
                                                                                                                                                                              The zebrafish has become                                                  found in fish. At the level of individ-
                                                                                                                                                                                                                                                        ual molecules, too, the creature re-
                                                                                                                                                                              one of biologists’ favorite            dysseus’ odyssey lasted 10

                                                                                                                                                                              pets. This is because zebra-
                                                                                                                                                                                                              O      years. Fate led him from island
                                                                                                                                                                                                              to island, he had to survive countless
                                                                                                                                                                                                                                                        sembles humans much more closely
                                                                                                                                                                                                                                                        than one would at first suspect. The
                                                                                                                                                                                                                                                        majority of genes found in humans
                                                                                                                                                                              fish go through all the steps   adventures and he lost all of his         also occur in fish, and there are
                                                                                                                                                                                                              companions before making it back          many indications that the functions
                                                                                                                                                                              from fertilization to becom-    home. A similar chain of events oc-       of zebrafish genes are often similar
                                                                                                                                                                                                              curs in a small fish. However, instead    or even identical to those of the cor-
                                                                                                                                                                              ing an independently viable     of the heroes of Homer’s famous           responding genes in humans. How-
                                                                                                                                                                              organism within just a few      saga, here it is cells that meander       ever, compared with humans, ze-
                                                                                                                                                                                                              around: in zebrafish that carry a ge-     brafish develop extremely rapidly.
                                                                                                                                                                              days. Processes that can take   netic defect, the precursors of the fu-   Just 24 hours after the egg is laid,
                                                                                                                                                                                                              ture germline cells no longer find        nearly all organs have begun to de-
                                                                                                                                                                              weeks in humans often re-       their way to the developing sex or-       velop: the brain folds in, the heart
                                                                                                                                                                              quire just a few hours in       gans. Instead, during development,        pumps blood through the veins and
                                                                                                                                                                                                              they wander aimlessly through the         the embryo begins to move. On the
                                                                                                                                                                              zebrafish. In the following,    body and run aground in strange lo-       third day of development, zebrafish
                                                                                                                                                                                                              cations. Because of the parallels be-     hatch and begin to swim. Finally, at
                                                                                                                                                                              RALF DAHM,     until recently   tween the odyssey of the Greek hero       five days, most cell types are differ-
                                                                                                                                                                              a staff member at the MAX       and the processes that take place in      entiated. The larvae don’t yet look
The zebrafish larvae depicted above are 40 hours old: The transparent eggshell offers a view of the already well-developed eyes.
However, it will take two to three months before the zebrafish are fully grown and have reached sexual maturity, as depicted below.                                                                           the zebrafish, the discoverers of this    like adult fish, but their organs al-
                                                                                                                                                                              PLANCK INSTITUTE      FOR       mutant christened it odysseus.            ready take up their functions. The
                                                                                                                                                                                                                 For biologists, genetic defects like   animals can see, smell, hear and find
                                                                                                                                                                              DEVELOPMENTAL BIOLOGY           the one just described are very inter-    food on their own.
                                                                                                                                                                              in Tübingen, describes the      esting. By analyzing the affected
                                                                                                                                                                                                                                                        A TRANSPARENT
                                                                                                                                                                                                              fish, they can learn a great deal
                                                                                                                                                                              advantages of the zebrafish                                               FISH IN THE MAKING
                                                                                                                                                                                                              about how genes control the devel-
                                                                                                                                                                                                              opment of the embryo and how, for         But not only is it possible to observe
                                                                                                                                                                              as a model organism.            example, the heart, the muscles, the      zebrafish development from the out-
                                                                                                                                                                                                              brain or, as here, the sex organs         side and watch how their bodies
                                                                                                                                      PHOTOS: MPI FOR DEVELOPMENTAL BIOLOGY

                                                                                                                                                                                                              form. But why do biologists study         slowly take on shape – it is even pos-
                                                                                                                                                                                                              zebrafish, of all beings, to under-       sible to see inside the developing fish.
                                                                                                                                                                                                              stand embryonic development? And          The embryos and young larvae are
                                                                                                                                                                                                              why a fish at all?                        almost completely transparent. Thus,
                                                                                                                                                                                                                 The idea goes back to American         it is possible to watch inside a living
                                                                                                                                                                                                              geneticist George Streisinger. In the     animal how the various organs form
                                                                                                                                                                                                              late 1970s, he was looking for a mod-     – even organs that lie deep within the

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FIRSTHAND KNOWLEDGE                                                                                                                                                                                                                                                  DEVELOPMENTAL BIOLOGY

            FROM EGG             TO      FISH    IN   JUST 48 HOURS
            Zebrafish embryos develop very rapidly compared with those of other vertebrates.                    involved in the relevant development      ses showed that odysseus mutants           fish exhibited a phenotype similar to            thus identified two molecules that
            Within just 4 hours, the fertilized egg cell, which rests atop a large yolk (above left),
            undergoes cell division and forms a cluster of several thousand, largely unspecialized
                                                                                                                process. The mutant odysseus was al-      carry a mutation in the CXCR4 gene.        the odysseus mutants: the germline               must act together for germline cells
            cells. Over the next 6 hours, these grow around the yolk. Within 10 hours after fertiliza-          so found in such a screen for fish        The result is that only part of the        cells again wandered aimlessly                   to reach the sex organs. However,
                                         tion, the head begins to show at the upper pole. After 18              with developmental defects.               corresponding protein is produced.         through the body and did not find                the distance that the germline cells
                                         hours, oval-shaped eye precursors are already discernable                 The germline cells – which produce     The truncated protein lacks impor-         their way to the sex organs. And yet             must cover during development is
                                         and the first muscles form in the body. A short time later,            the sperm and egg cells – have a spe-     tant parts that are crucial for its        a further experiment suggested that              very long, and it seems unlikely that
                                         the tail begins to extend.
                                                                                                                cial status: they form the cells that     function – the fish develops as if this    the SDF-1 signal plays a key role in             a single pair of signaling molecules
                                              After 24 hours, most of the organs are in place, the heart        pass genetic information from one         protein were completely missing. In        the cells’ success in finding their              can direct them the entire way.
                                              begins to beat and the fish is already moving inside the egg.
                                              Two days after fertilization, most organs have formed and
                                                                                                                generation to the next. From a bio-       parallel with the studies on odysseus,     goal. If, through a genetic trick,               Modulating factors presumably also
                                              taken up their functions. On the third day, the larvae hatch      logical standpoint, the germline is all   researchers led by Erez Raz at the         SDF-1 is no longer produced in the               play an important role in ensuring
                                              and swim their first short distances. After five days, they       that remains of an individual. This       Max Planck Institute for Biophysical       sex organs, but somewhere else in                the cells’ accurate navigation. We do
                                              have largely consumed their yolk supply and independently         characteristic makes the germline         Chemistry in Göttingen were able to        the body instead, the germline cells             not yet know what factors these are
                                              go in search of food.                                             cells especially interesting. Their im-   show that CXCR4 is needed to put           migrate to this new place and collect            and how they interact with the
                                              Despite the rapid embryonic development, it still takes two or    portant function in the succession of     germline cells in zebrafish on the         there. These zebrafish experiments               CXCR4/SDF-1 pair.                  ®
                                              three months before the zebrafish is sexually mature and can      generations is also reflected in their    right path.
                                              reproduce. Measuring just three to five centimeters in length,
                                              adult zebrafish are quite small. The eggs and the young larvae
                                                                                                                formation during embryonic devel-            The CXCR4 protein was already
                                              are even so tiny (less than one millimeter) that dozens of        opment. They break away from the          known before. It belongs to a family
                                              them can be kept in small petri dishes. Its small embryo size     other cells of the embryo early on        of receptors whose members play a
                                              has a further advantage: individual embryos can be placed in-     and undergo a special program.            role, for example, in the migration of
                                              to tiny reaction tubes. So-called microtiter plates that hold        In mice, for instance, the precur-     white blood cells. In mice, CXCR4 is
                                              nearly a hundred of these tubes can then be used in auto-
                                              mated processes to test the impact of entire libraries of small
                                                                                                                sors of germ cells arise in the em-       also involved in the targeted growth
                                              molecules within the organism – be it to identify molecules       bryo, but then they migrate out of        of nerve fibers and blood vessels.
                                              that can cause deformities in normal fish or to find sub-         the embryo and into the yolk sac.         However, a function relating to the
                                              stances that can mitigate a genetic defect.                       Only about three weeks later can          migration of germline cells had nev-
                                                                                                                they be found in the embryo again,        er been observed before. The studies
                                                                                                                at the location where the sex organs      on the mutant odysseus zebrafish fi-
          body. Scientists can observe how, for                  how genes control these processes.             form during development. In ze-           nally provided the connection: in the      The striped pattern of the zebrafish is produced by three types of cells
                                                                                                                                                                                                     with different pigments. Mutations of these cells can lead to different patterns.
          example, different regions of the                      If, however, one of the genes is de-           brafish, too, the precursors of the       migrating germline cells, CXCR4 is
          brain develop, the muscles and the                     fective, scientists have the opportu-          germline cells have to migrate from       the receptor that detects the direc-
          digestive apparatus form, the blood                    nity to understand how it functions.           where they are born to the future sex     tional signal. If it doesn’t function,
          vessels grow, or the heart takes on its                This idea stems from a simple under-           organs. How do the precursor cells        the cells get lost even if the signal is
          shape and begins to beat.                              lying logic: genetic defects (muta-            find the way to their destination         present. This would be similar to a
             In addition, the behavior of indi-                  tions) result in a gene no longer be-          when migrating through the body?          person traveling through an unfa-
          vidual cells can be monitored much                     ing able to perform its function dur-          And what signal directs the migrat-       miliar city looking for a particular
          more easily in zebrafish than in oth-                  ing development. Thus, organisms in            ing cells? Which receptor in the cells    address and not being able to read
          er vertebrates. The migration of germ                  which a specific gene is mutated ex-           receives this signal? Until recently,     the street signs.
          line cells in the odysseus mutant are                  hibit characteristic developmental             these were the great unsolved ques-
                                                                                                                                                          A SIGNPOST
          a good example. Moreover, it is pos-                   abnormalities and have a different             tions about the development of
                                                                                                                                                          IS NOT ENOUGH
          sible to see how the nerve cell                        appearance (phenotype), depending              germline cells.
          processes grow and come into con-                      on what role the intact gene normal-              Now, two studies on zebrafish          That solved the first part of the mys-
          tact with one another, and how a                       ly plays.                                      have nearly solved this puzzle. In        tery. And the researchers were also
          functioning nervous system devel-                                                                     one of them, the odysseus mutant          able to identify the signal that the
                                                                 GENETICISTS LEARN
          ops. Because of this, in the early                                                                    with its aberrant germline cells          CXCR4 receptor detects in the
                                                                 FROM MISTAKES
          1990s, more and more developmen-                                                                      played a major role. Investigations       germline cells. In addition, it was
          tal biologists recognized the poten-                   Mutant phenotypes thus allow re-               by Holger Knaut in Nobel laureate         previously known from studies on
          tial of the zebrafish and carried                      searchers to draw conclusions about            Christiane Nüsslein-Volhard’s group       mice that CXCR4 interacts with the
          Streisinger’s idea further. Among                      the normal function of the mutated             at the Max Planck Institute for De-       molecule SDF-1. When the scientists
          other things, they were looking for                    gene. For example, if heart develop-           velopmental Biology in Tübingen           in Tübingen and Göttingen looked at
          fish with genetic defects. Their aim                   ment is disrupted in a mutant, we              showed that the precursors of the         the pattern of the SDF-1 activity in
          was to identify which genes play an                    know that the mutated gene normally            germline cells in this fish can, in       zebrafish embryos, they noticed that
          important role in the development of                   plays a role in forming the heart. So          fact, still migrate, but they can no      SDF-1 maps out the route for the mi-
          vertebrates.                                           by seeking particular mutants with             longer find their target tissue. In-      grating germline cells. When they
             Studying an organism’s normal                       specific developmental defects, it is          stead, they wander aimlessly              deactivated SDF-1 in the embryo,
          development tells us nothing about                     possible to find many of the genes             through the body. Molecular analy-        this guiding pattern was lost and the

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FIRSTHAND KNOWLEDGE                                                                                                                                                                                                                               DEVELOPMENTAL BIOLOGY

                                                                     Up to 200
                                                              zebrafish larvae
                                                               splash around                                                                                                                                                         OF MACHOS              AND        CREAM PUFFS
                                                              in a petri dish.                                                                                                                                                       Many genes that play an important role in development
             The search for zebrafish mutants                                                                             Despite some very     artificially and, via homologous re-      whether one gene can compensate
                                                                                                                                                                                                                                     were named after what an animal looks like when that
          began with the aim of gaining fun-                                                                           promising     achieve-   combination, put in place of the in-      for the loss of another.                   gene is defective. Every now and then, these mutants are
          damental insights into embryonic                                                                            ments, identifying mu-    tact gene in embryonic stem cells.           Nevertheless, compared with the         given an entirely unscientific name. A rather small, squat
          development in vertebrates. But be-                                                                       tated genes in zebrafish    The genetically modified stem cells       knockout, the knockdown has one            Drosophila (fruit fly) mutant, for instance, is called
          yond that, the mutants can serve as                consists of nearly                                   is still a very laborious     are injected into early mouse em-         key disadvantage: using morpholinos        knirps (German for “tot”). A number of fly mutants that
                                                                                                                                                                                                                                     always die out in the third generation were named after
          models for human diseases. One such                27,000 amino acid                                task. To date, the genetic de-    bryos and integrate into the embryo       to deactivate genes is truly effective
                                                                                                                                                                                                                                     fallen royal dynasties: staufen, tudor, valois and vasa.
          mutant is pickwick, which was dis-                 building blocks. By way of               fects underlying an observed mutant       during development. At present,           only during the early stages of devel-     Cactus larvae have more bristles and windbeutel (“cream
          covered in the early 1990s in the                  comparison, rhodopsin, the protein       phenotype have been found for only        knockouts cannot be performed in          opment, as morpholinos become in-          puff”) larvae are hollow, as they are missing their inter-
          laboratories of Wolfgang Driever and               that enables us to see, comprises on-    about 400 mutants. And there are          zebrafish because there are no em-        creasingly diluted as the organism         nal organs. Flies with a mutated cucumber gene lay eggs
          Mark Fishman in Boston. With the                   ly 348 amino acids. Analyses of the      still countless mutants whose pheno-      bryonic stem cell lines and the           grows during development. Their ef-        that actually resemble the tubular fruit. Another mutant
                                                                                                                                                                                                                                     made its discoverers so happy – because it proved so en-
          exception of a heart dysfunction,                  titin DNA sequence showed that           types have been described in detail,      method of homologous recombina-           fect becomes weaker and weaker in
                                                                                                                                                                                                                                     lightening for their research – they called it simply toll
          pickwick mutants exhibit no defects                pickwick mutants indeed carry a          but for which the affected genes re-      tion has not been established.            later developmental stages. Processes      (“great”).
          in their development. And in the be-               mutation in the titin gene. Intrigu-     main unknown. Recently, the ze-              Instead, an alternative technique      that occur later in development, or
                                                                                                                                                                                                                                     Assigning creative names has long been standard prac-
          ginning, even the heart forms nor-                 ingly, the mutation affects a region     brafish genome project, which aims        was conceived a few years ago to          that take place only as the organism       tice among fly researchers, but in the past few years, ze-
          mally, at least as far as can be                   of the protein that is produced only     to decode the sequence of all of the      deactivate specific zebrafish genes:      ages, are difficult or even impossible     brafish have followed suit. Faulpelz (“lazy bones”) is the
          judged by looking at it from the out-              in heart muscle cells and is missing     DNA building blocks in the zebrafish      the knockdown. In this technique,         to investigate using morpholinos.          name of a mutant that moves less than normal fish. Ma-
          side. However, from its first beat on,             in titin variants produced by other      genome, has been a great help in          chemicals known as morpholinos are           For this reason, a second tech-         chos don’t react when they are shoved, obelix has broad-
                                                                                                                                                                                                                                     er stripes, and zwangsjacke (“straitjacket”) hatches from
          its contractions are very weak and it              muscle cells. That explains why the      searching for these genes. This joint     injected into the fertilized egg. Mor-    nique has been applied to zebrafish:
                                                                                                                                                                                                                                     the egg more slowly than its normal conspecifics. Ze-
          pumps hardly any blood through the                 skeletal musculature in pickwick de-     endeavor was launched in 2001 by          pholinos are artificially manufac-        TILLING. Though methodologically           brafish mutants with malformed ears (fish have only the
          body. Later, it swells up and the                  velops completely normally.              the British Sanger Institute, together    tured oligonucleotides and thus be-       different from the classic knockout        inner part of the ear, no auricle) have also inspired cre-
          mutant dies of heart failure. These                                                         with groups in Germany, the Nether-       long, chemically speaking, to the         performed on mice, this technique          ative names: spock has pointy ears, van Gogh has smaller
                                                             A MUTATION THAT GOES                                                                                                                                                    ones and eselsohr (“dog-ear”) mutants have a crease in
          observations suggested that a closer                                                        lands and the US. With a total of 1.5     same group as DNA and RNA mole-           also results in animals in which a
                                                             RIGHT TO THE HEART                                                                                                                                                      their ears. Also on the long list of imaginative monikers
          look at the cells that form the heart                                                       billion building blocks, the zebrafish    cules. They are likewise made up of       specific gene is mutated. In contrast
                                                                                                                                                                                                                                     is faust, a mutant whose breast actually holds two beat-
          muscle was in order.                               In heart muscle cells, titin is impor-   genome is about half the size of          four building blocks: the bases A, C,     to knockout mice, however, TILLING         ing hearts, and liebeskummer (“lovesickness”), a fish
             Normal heart muscle cells contain               tant for the correct arrangement and     man’s. For the zebrafish genome,          G and T. However, the bases in mor-       mutations are induced randomly             with a tiny stomach and a big heart.
          countless protein filaments that be-               anchoring of the protein filaments. It   however, the decoding will be easier      pholinos are joined by a molecule         rather than specifically. Thus, the
          gin at opposite ends of the cell and               thus plays an essential role in heart    because the researchers can draw on       that does not normally occur in cells.    offspring of the mutagenized fish
          interlock like two brushes whose                   muscle contractions. From a medical      the experience and methods from the       The cells therefore have no enzymes       must subsequently be systematically      are working on a three-dimensional
          bristles are pressed together. When                perspective, the pickwick mutant is      human genome project. The project         to break down morpholinos. Once           screened for mutations in the gene       atlas of brain development to better
          the heart beats, these filaments mi-               particularly interesting because its     should soon be completed, and then        they are injected into the egg cell,      of interest. To find mutations, many     understand, at the level of individual
          grate past one another to the center               heart resembles that of patients who     the genome sequence will be freely        the morpholinos remain stable and         such offspring must be examined.         cells, how the brain gradually takes
          of the cell. This brings the ends of the           suffer from what is known as dilata-     accessible on the Internet.               are inherited by the newly formed         However, because laboratory ze-          on its form during development –
          cell closer together – the cell con-               tive cardiomyopathy. In both hu-            In the meantime, the zebrafish has     cells during development.                 brafish are much easier to keep than     and showing it at higher resolution
          tracts. If the filaments migrate away              mans and zebrafish mutants, this         found a new niche: decoding the                                                     mice, TILLING is, on the whole, not      than has ever been done for any ver-
                                                                                                                                                MORPHOLINOS AS
          from each other again, it relaxes.                 heart disease is characterized by thin   functions of previously unknown                                                     as laborious as producing knockout       tebrate. Soon, the zebrafish will be-
                                                                                                                                                “WHITE-OUT” FOR GENES
          Fishman’s group discovered that the                walls, swelling and weak contrac-        genes predicted by the genome pro-                                                  mice. And compared with the knock-       long to the handful of organisms
          heart muscle cells in pickwick mu-                 tions of the heart. Medical profes-      jects. To track them down, they can       The sequence of bases in a morpholi-      down method, TILLING has the ad-         about which more is known than any
          tants contain far fewer of these pro-              sionals estimate that, in approxi-       be specifically deactivated in ze-        no is chosen to precisely match part      vantage that the mutations are per-      other – in some respects, even more
          tein filaments than normal cells.                  mately one third of patients, these      brafish. Until recently, a gene           of the mRNA of a targeted gene. In        manent and their effects can be ob-      than is known about humans.         ●
          Consequently, the cells contract very              pathological changes in the heart        knockout was possible only in             the cell, the injected morpholino         served throughout development as
          weakly. They are thin, and the heart               have genetic causes. To date, howev-     mice. In this procedure, a defective      binds to this mRNA and prevents it        well as in adulthood and aging.
                                                                                                                                                                                                                                                        DR. RALF DAHM, born in
          muscle swells up and is limp.                      er, they have succeeded in identify-     variant of the gene is produced           from being translated into the corre-        Zebrafish research has enormous                            1970, studied biology
             What interested the researchers                 ing the corresponding defective                                                    sponding protein. For the cell that       potential. This is evidenced, for in-                         and biochemistry at
          was why the arrangement of protein                 genes in only a few cases. In parallel                                             lacks the protein, it is as if the re-    stance, by the fact that the European                         Saarland University,
          filaments that is typical for muscle               with investigations of the pickwick      Not yet able to swim,                     spective gene were absent. The effect     Commission is sponsoring a joint                              the University of
                                                                                                      this two-day-old larva still                                                                                                                      Tübingen and the Med-
          cells can’t form in pickwick. They                 mutant, a consortium of clinical re-                                               is thus very similar to that of a         project of 15 European research in-
                                                                                                      feeds on its yolk supply.                                                                                                                         ical Sciences Institute
          mapped the mutation to the genome                  search groups from Germany, Aus-                                                   mouse knockout. It’s effect, however,     stitutes, under the direction of the                          at Scotland’s University
          and discovered that it lies in a region            tralia, the UK and the US analyzed                                                 is visible already in the injected fish   Max Planck Institute for Develop-                             of Dundee, where
          that contains a candidate gene. The                the human titin gene. This study did,                                              themselves, not only in subsequent        mental Biology in Tübingen, to the       he also did his doctoral studies. Thereafter,
          gene in question codes for a protein               indeed, identify patients that carry a                                             generations, as is the case for knock-    tune of 12 million euros since 2004.     Ralf Dahm worked at the Max Planck
                                                                                                                                                                                                                                   Institute for Developmental Biology in
          that was already known from heart                  mutation in the titin gene. This                                                   out mice. In addition, the morpholi-      Using the zebrafish, the project aims
                                                                                                                                                                                                                                   Tübingen for six and a half years.
          and skeletal muscles: titin – the                  example shows just how medi-                                                       nos make it possible to deactivate        to explore the development of verte-     In July 2005, he joined the Center for
          largest protein ever identified in a               cine can profit from the findings                                                  more than one gene at a time, and         brates, as well as human hereditary      Brain Research at the Medical University
          vertebrate. A single titin protein                 of zebrafish research.                                                             thus to test how genes interact and       diseases. Moreover, the researchers      of Vienna as a senior research scientist.

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