1997 Oxford University Press
Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
1755–1760
Zebrafish genetics and its implications for understanding vertebrate development P. W. Ingham Developmental Genetics Programme, The Krebs Institute, University of Sheffield, Sheffield S10 2TN, UK Received May 22, 1997
The identification of genes via their mutant phenotypes is the most direct way of dissecting the molecular basis of developmental processes. While this approach has been extremely powerful in invertebrates such as Drosophila and Caenorhabditis elegans, it has until recently been lacking in vertebrates. Now two landmark studies by the groups of Nüsslein-Volhard and Driever have for the first time provided the basis for a comprehensive genetic dissection of vertebrate development using the teleost fish, Danio rerio. The mutations identified in these screens look set to revolutionise our understanding of vertebrate development.
INTRODUCTION In recent years the application of genetic analysis has transformed our understanding of developmental biology in a way hardly imaginable little more than a decade ago. Much of this is due to the success of the strategy adopted by Nüsslein-Volhard and Wieschaus (1) in their effort to identify all of the genes controlling embryonic development of the fruit-fly Drosophila. Their approach was simple—a screen for embryonic lethal mutations in the F3 generation of mutagenised flies—but the results have been profound, not only illuminating our understanding of Drosophila embryogenesis, but contributing many new insights into the development of all animals, from insects to humans. A key aspect of the Nüsslein-Volhard and Wieschaus screening strategy was its focus on mutations with clearly defined phenotypes; in this way they aimed to identify only those genes that have a unique role in a specific developmental process. And to be confident that all such genes were isolated, they pursued the screens to saturation, that is until the only mutations recovered were alleles of genes identified earlier in the screen (2–4). One of the most important findings of these screens was the discovery of particular classes of mutants with common phenotypic traits. We now know that, in many cases, these mutations identify genes encoding elements of specific developmental pathways, but it is precisely through their recovery and analysis that the elucidation of these pathways has been possible. Examples of such phenotypic classes include the segment polarity mutations, many of which have been found to encode components of the Wingless (5) and Hedgehog (6) signal transduction pathways, as well as the Dorsal group of mutations (7), which identify genes involved in a signalling pathway that culminates in the activation of the Dorsal protein, a homologue of the mammalian transcription factor NFκ-B. It is, of course, through the discovery of homologues of these and many other genes that the results of the Drosophila screens
have impacted so dramatically upon our understanding of vertebrate development. Not only have sequence homologies provided a means of ‘fishing’ for developmental genes in vertebrate genomes but, more significantly, such homologous genes have in many cases been found to regulate remarkably similar processes in the development of flies and vertebrates alike (6,8). While these discoveries have encouraged the use of Drosophila as an experimental paradigm for many aspects of vertebrate development, it is clear that there are limitations to this approach. Neural crest, for instance, is a vertebrate invention that has no counterpart in Drosophila from which the molecular basis of its development could be extrapolated. Even where analogous structures or systems do exist, the similarities can often be superficial. The vertebrate heart and circulatory system, for example, differ significantly in form and function from their more primitive equivalents in insects; although one of the earliest indications of the heart in the vertebrate embryo is the activation of the Nkx2.5 gene (9)—the homologue of tinman, a gene essential for Drosophila heart muscle development (10)—there are many aspects of cardio-vascular development that are unique to vertebrates. THE ZEBRAFISH MUTANT SCREENS Given these considerations, the case for emulating the saturation mutagenesis approach in a vertebrate seemed compelling and at the end of 1996 the fruits of the first systematic mutational screens of any vertebrate species were reported in a special issue of the journal Development by the groups of Nüsslein-Volhard and Driever (11,12). The choice of the zebrafish for such an approach was influenced by many factors, not least of which is the possibility it affords of breeding large numbers of animals in a relatively small space and in a relatively short time. This is essential since a conventional screen for recessive lethals in a diploid requires three generations, and to approach saturation one
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1756 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review
Figure 1. Living zebrafish embryos at successively later stages of development. (A) 8 cell stage, 1.25 hpf (hours post fertilisation). (B) Blastoderm stage, 4 hpf. (C) Embryo commencing gastrulation 6 hpf. (D) Embryo at completion of the extension of the primary axis, 10 hpf. (E) 5 somite stage, 11.5 hpf. (F) 14 somite stage, 16 hpf. (G) Embryo 24 hpf. Abbreviations: b, brain; e, ear; n, notochord; s, somites; t, neural tube; y, eye. (H) Free swimming larva, 120 hpf. Abbreviations: a, gill arches; e, ear; g, gut; h, heart; l, liver; n, nasal pit.
must score enough mutagenised genomes to be sure of hitting each gene more than once. With a generation time of 10–12 weeks, animals homozygous for a mutation can be identified within 9 months of its induction, and with an average single locus induced mutation rate of the order of 10–3, a screen of several thousand genomes should approach saturation. The identification of mutations in the zebrafish is greatly facilitated by another important property of this species, the complete transparency of its embryos (Fig. 1). Simply by observing the living embryo under a dissecting microscope it is possible to identify structures such as the notochord, neural tube, heart, cerebellum, olfactory pit and dorsal aorta within 24 h of fertilisation of the egg; after 5 days of development, organs such as the tail artery, intestine, stomach and liver, as well as the mouth, eyes and muscles are all clearly visible (13). Accordingly, with the exception of one class [mutations affecting retino-tectal projections which were detected by filling axons (14)] all of the mutations recovered in the screens were identified exclusively on the basis of morphological or behavioural defects observed in living embryos (see Fig. 2 for examples). Together, the two groups identified a total of 6647 mutations from amongst 6194
mutagenised genomes, a frequency approaching 1.1 mutations per genome sampled (11,12). So efficient was the process that many more mutants were generated than could be handled and both groups took the decision to discard one large class of mutations, characterised as having ‘general’ abnormalities. The rationale for this cull was that such mutations are too pleiotropic to be useful in dissecting specific developmental pathways. While this judgement is supported to some extent by previous experience with Drosophila, it is likely that many of the mutations overlooked in this way would ultimately have turned out to be informative. Most likely, such mutations will be recovered again when more directed screening strategies, for instance, using changes in the early patterns of gene expression as a phenotype, are pursued. The elimination of mutations with ‘general’ abnormalities left ∼30% of the original collection, all of which exhibit relatively specific defects. These mutations have been grouped into classes on the basis of their phenotypes and mutations within a given class tested for complementation. This analysis is still not complete; in particular, extensive complementation testing between mutations isolated by the two different groups has not been
1757 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1757 Nucleic Acids Research, 22,10 No. 1
Figure 2. Wild-type (A) and mutant embryos (B and C) 60 hpf. (B) Embryo homozygous for the cyclops mutation. This is easily recognisable by its reduced eyes which actually fuse across the midline to form a single eye, hence the name. A similar phenotype is exhibited by the one eyed pinhead (oep) mutation. (C) Embryo homozygous for the floating head mutation. Note the drastic reduction in the length of the body axis, due to the failure of the development of the notochord (the notochord is clearly visible in the wild-type and cyclops embryos).
undertaken. However, from the results obtained to date it is clear that the screens have only reached ∼50% saturation, with fewer than half of the genes so far being identified by more than one allele. The phenotypic classes cover most aspects of embryogenesis, from mutants affecting the cell movements of gastrulation to those disrupting primary axis formation, axial mesoderm specification, notochord and somite differentiation and the development of the brain, ears, eyes and jaw as well as of organs such as the kidney, liver and cardio-vascular system. In the following sections I will consider the ways in which these mutations are adding a new dimension to our understanding of vertebrate development by reference to the analysis of a few selected classes. PATTERNING THE PRIMARY EMBRYONIC AXES One of the major puzzles in vertebrate developmental biology has long been how pattern is established along the primary embryonic axes. In recent years a number of candidate molecules have been implicated in this process, largely on the basis of studies in the amphibian, Xenopus laevis. The ability to assay the activity of gene products by injecting synthetic mRNA into newly fertilised Xenopus eggs has proven very powerful in the search for such axis patterning molecules. However, a significant limitation of this approach is that it can only establish the sufficiency and not the necessity for a given factor. Examples of such factors are the secreted proteins Follistatin, Noggin and Chordin (15–17), each of which has been shown to promote the formation of dorsal mesoderm and neurectoderm at the expense of ventral structures. All three proteins have been proposed to act by antagonising the effects of two members of the TGFβ superfamily of secreted proteins, BMP2 and BMP4, both of which are expressed on the ventral side of the developing embryo and which, when
overexpressed, transform cells fated to become neurectoderm and dorsal mesoderm towards more ventral fates. While Noggin, Follistatin and Chordin are all expressed at the right time and in the right place to play a role in promoting dorsal patterning, it has remained a moot point, in the absence of loss-of-function analysis, as to which, if any, of these factors is actually required for this process. Given this background, some of the most intriguing mutations to come out of the fish screens are those displaying defects in dorso-ventral patterning (18,19). In embryos homozygous for the dino mutation, dorsal cells express ventral fates as early as the gastrula stage, whereas in swirl mutant embryos, ventral cells adopt dorsal fates. These phenotypes suggest that the two genes may respectively encode dorsalising and ventralising activities, and consistent with this, the dino phenotype can be suppressed by inactivation of BMP4 signalling while swirl is completely epistatic to dino and can itself be partially rescued by injection of BMP4 mRNA (20). The analysis of these two mutations thus provides strong support for the Xenopus model of patterning along the dorso-ventral axis by opposing gradients of BMP and BMP-antagonist activities, prompting Schulte Merker and colleagues to explore the possibility that dino might itself encode one of the three dorsalising factors identified in Xenopus. Using RFLP analysis, these authors have now demonstrated that dino does indeed correspond to the zebrafish chordin gene (21); thus the dino mutation provides the first definitive evidence for a dorso-ventral organising activity in a vertebrate embryo (21). While establishing the biological significance of Chordin, this important finding raises questions about the contributions of Noggin and Follistatin, questions which analysis of the other mutations in this class should help to resolve.
1758 Human Molecular Genetics, 1997, Vol. 6, No. 10 Review AXIAL MESODERM AND MIDLINE SIGNALLING Once the primary axes of the vertebrate embryo have been established, the axial mesoderm assumes a central role in the patterning of a number of key tissues including the brain, neural tube and somites. The principal evidence for this has come from studies in chick in which the inducing activities of axial mesodermal structures have been assayed by grafting them to ectopic locations (22); more recently, the secreted protein encoded by the Sonic hedgehog gene has been identified as the primary mediator of axial mesoderm inducing activity (6). Defects in midline signalling have important clinical consequences resulting in common congenital defects in humans, such as holo pros-encephaly. Yet despite this, genetic studies of the development and function of axial mesoderm in the mouse have been somewhat limited, focusing on the classical brachyury mutation and, more recently, on mutations generated in genes expressed in axial mesoderm such as Lim1, HNF3β and Shh itself (23–25). The repertoire of genes implicated in the development and function of the axial mesoderm has now been significantly expanded by the zebrafish screens. Some mutations, such as one eyed pinhead (oep) (26,27) affect only the most anterior axial mesoderm, the prechordal plate, leaving the more posterior axial derivative, the notochord, unaffected. Failure of the prechordal plate to develop leads to a failure in the induction of ventral brain structures and cyclopia (Fig. 2), presumably due to the absence of Shh signalling. The majority of mutations affecting axial mesoderm, however, identify genes required for the development of the notochord (28,29) (Fig. 2). Two of these, no tail (ntl) and floating head (flh) had previously been described, both at the genetic and the molecular level. ntl encodes the zebrafish homologue of the T-box protein encoded by Brachyury (30), whilst flh encodes a homologue of the Xenopus homeodomain protein XNot1 (31). Amongst the other mutations isolated, several, including bozozok, momo, doc and gnome, have phenotypes similar to those of ntl or flh, lacking differentiated notochords and showing defects in the induction of floorplate (the most ventral cell type in the neural tube), and of muscle pioneer cells, a subset of muscle cells known to be dependent upon inductive signals from the notochord (32,33). Since none of these genes have yet been cloned it is not known where they are expressed; however, it seems likely that they will define a pathway of activities required for the establishment and differentiation of axial mesoderm structures. By contrast, embryos homozygous for a second group of mutations have normal prechordal plate and notochord yet nevertheless show defects in neural tube and somite patterning, suggesting that they somehow disrupt signalling by the axial mesoderm (34). Amongst these, the strongest phenotypes are shown by you-too (yot) and chameleon (con), both of which result in a reduction in secondary motor neurons, mild cyclopia and absence of muscle pioneer cells. Interestingly, by generating embryos that are genetically mosaic—a technique that in the fish is relatively simple—yot activity has been found to be required not in the notochord or prechordal plate, but rather in the paraxial mesoderm (34), implying that in this case the gene functions in the response of cells to the midline derived inducing signal. Although similar mosaic analyses have yet to be performed for the other mutations in this group, it seems likely that at least some of them will identify components of the pathway that transduces
the inducing signal emanating from the axial mesoderm. This signal, is of course expected to be encoded by Shh, so it is no surprise that RFLP analysis has shown that one of these genes, sonic you (syu), is indeed the zebrafish shh (P. Haffter, F. van Eeden, H, Schauerte and C. Nüsslein-Volhard, personal communication). What is surprising is that the syu phenotype is somewhat weaker than that of either con or yot (34). One possible explanation for this is an unanticipated yet characteristic feature of the zebrafish genome, namely the presence of extensive gene duplications. Two closely related homologues of shh have been found to be expressed in axial midline structures in the zebrafish (33,35), either one of which could compensate for the loss of shh activity in the syu mutation. This serves to highlight one of the potential shortcomings of the zebrafish as a model for genetic analysis; clearly, in order to extrapolate the analysis of fish development to other vertebrates it will be necessary to identify mutations in all cognates of any given gene—and there is the obvious problem that in some cases, the phenotypes of these mutations may be so subtle as to go undetected. ORGANOGENESIS: THE CARDIO-VASCULAR SYSTEM The facility with which the heart can be identified and observed in living embryos, together with the lack of dependence of embryonic development on a functioning circulatory system, has made the zebrafish a particularly attractive model for the analysis of cardio-vascular development. The heart primordia can be clearly identified at ∼16 h of development through the restricted expression of the Nkx2.5 gene in two discrete domains flanking the midline, just anterior to the notochord (36); these domains fuse ∼3 h later to form the primitive heart tube. Although the fish heart is not separated into left and right chambers, it nevertheless undergoes the same rightward looping that marks the onset of chamber formation in air breathing vertebrates; this process, which occurs at ∼36 h is preceded by a transient inflexion in the opposite direction ∼12 h earlier, which can be visualised by the asymmetric expression of BMP4 in the developing heart tube. A number of mutations have been found to affect these early phases of heart development either reversing or randomising its left/right asymmetry. These include mutations affecting development of the notochord and/or floorplate such as flh, ntl, cyclops (37). The further analysis of these and other mutants may well yield insights into the basis of a number of congenital abnormalities thought to arise through the perturbation of cardiac looping. Other mutations have been identified which affect later aspects of heart development including tubulogenesis, chamber generation and orientation, valve formulation and concentric growth (38). To date, the most extensively analysed of these is the cloche mutation (39). In animals homozygous for cloche the endocardium is completely absent yet the myocardial tube forms and is divided into two chambers; however, the subsequent development of these is abnormal, the ventricle being reduced in size whilst the walls of the atrium are distended, indicating that the endocardium is required for maturation of the myocardium. In embryos homozygous for pandora the heart lacks the ventricle (38), a situation reminiscent of the clinical condition hypo plastic chambers. Embryos homozygous for miles apart and bonnie and clyde exhibit cardia bifida, each heart having two chambers and beating independently though devoid of blood flow since there is no connection to the dorsal aorta (38).
1759 Human Molecular Genetics, 1997,1994, Vol. Vol. 6, No. Review 1759 Nucleic Acids Research, 22,10 No. 1 The majority of the heart mutations isolated affect function rather than structure (38). In reggae mutants for instance, the normal sequential atrium and ventricular contractions are blocked and instead a wave of contraction initiates in the atrium but is rarely propagated, a situation resembling the clinical condition sinus exit block. Other functional mutants include tremblor which exhibits chaotic uncoordinated contractions of the atrium, silent partner, in which atrial contraction fails to lead to ventricular activity and island beat, in which individual cardio-myocytes contract independently but never trigger a contraction. Given that arhythmias are the most common adult human heart condition, the analysis of these and other similar mutations may also have important clinical implications.
FUTURE PROSPECTS The identification of so many mutations affecting zebrafish embryogenesis represents a quantum leap in our capacity to unravel the mechanisms underlying vertebrate development. Yet before the full potential of this vast resource can be realised a number of challenges remain to be met. Chief amongst these is the problem of cloning the genes identified by these mutations, for without their molecular characterisation, their usefulness remains limited. One solution to this problem—the candidate gene approach—has already proven successful in a number of cases, where well informed guesses have linked mutant phenotypes to known genes. So far, however, this approach has been limited to the identification of mutations with genes whose expression and function has already been extensively characterised in other systems (as described above). If the fruits of the screens are to inform rather than simply confirm our understanding of gene function, ways must be developed of linking mutations to genes independently of a knowledge of their function. In order to achieve this, a high resolution genetic map is a necessity. Progress towards the generation of such a map has been impressive; as little as 4 years ago, no two loci had been shown to be linked in the zebrafish genome yet by 1996, Postlethwait and colleagues (40) had generated a consolidated linkage map comprising 652 PCR-based markers with an average density of 1 per 4–3 cM. Locating mutations on this map is greatly facilitated by the ability to perform half tetrad analysis in zebrafish (41). Blocking the second meiotic division allows gene-centromere distances to be calculated whilst at the same time enabling the gene to be mapped to a linkage group through the use of centromere specific PCR based markers (40). At ∼600 kb per centiMorgan any given mutation can currently be located on average, to within 1200 kb of a molecular marker. As the density of such markers increases— with the addition of STSs, ESTs and cloned sequences identified in other ways—the molecular characterisation of genes identified by mutations should become relatively routine. The future for the zebrafish looks bright indeed!
ACKNOWLEDGEMENTS I am grateful to Dr Linda Parsons for her critical reading of the manuscript and to Dr Jörg Odenthal for providing the photographs for Figures 1 and 2.
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