Gilbert, Michael J. Eleventh edition. The ninth edition has been substantially revised and reorganised to reflect the very latest advances in the subject.
With an emphasis throughout on the evidence underpinning the main conclusions, this book is suitable as the key text for both introductory and more advanced courses in developmental biology. Contains expanded treatment of mammalian fertilization, the heart and stem cells. The book features exceptionally clear two-color illustrations, and is designed for use in both undergraduate and graduate level courses.
The book is especially noteworthy for its treatment of development in model organisms, whose contributions to developmental biology were recognized in the Nobel Prize for physiology and medicine.
Developmental Biology, Sixth Edition explores and synthesizes the organismal, cellular, and molecular aspects of animal development, and expands its coverage of the medical, environmental, and evolutionary aspects of developmental biology. Shorter than the previous edition by some pages deleted material available at www. Tyler and Ronald N. Kozlowski of the University of Maine. In Molecular Methods in Developmental Biology: Xenopus and Zebrafish, Matthew Guille assembles a hands-on collection of basic and essential molecular and embryological techniques for studying Xenopus and zebrafish.
Easily reproducible and designed to succeed, these detailed methods include cellular techniques, techniques for the quantitative and spatial analysis of mRNA and proteins, and techniques for the expression of gene products in embryos. More specialized methods enable users to analyze promoters and transcription factors during early development, and include gel shift assays, as well as in vitro and in vivo footprinting.
Wherever possible, these experimental approaches are applied to both Xenopus and zebrafish. Molecular Methods in Developmental Biology: Xenopus and Zebrafish affords newcomers rapid access to a wide variety of key techniques in developmental research, and offers experienced investigators both new techniques from experts who have fine-tuned them for best results, and a plethora of time-saving tips.
State-of-the-art and readily reproducible, these powerful methods constitute today's gold-standard laboratory manual for understanding the interactions responsible for development. Essential Developmental Biology is a comprehensive, richly illustrated introduction to all aspects of developmental biology. Written in a clear and accessible style, the third edition of this popular textbook has been expanded and updated In addition, an accompanying website provides instructional materials for both student and lecturer use, including animated developmental processes, a photo gallery of selected model organisms, and all artwork in downloadable format.
With an emphasis throughout on the evidence underpinning the main conclusions, this book is an essential text for both introductory and more advanced courses in developmental biology. Reviews of the Second Edition: "The second edition is a must have for anyone interested in development biology.
New findings in hot fields such as stem cells, regeneration, and aging should make it attractive to a wide readership. Overall, the book is concise, well structured, and illustrated. I can highly recommend it. This effort is no exception. Every student of developmental biology should experience his holistic yet analytical view of the subject. This thoroughly revised 4th edition offers both clear descriptions and explanations of human embryonic development based on all the most up-to-date scientific discoveries and understanding.
Particular attention is paid to the fundamental aspects of molecular mechanisms in development, introducing you to major families of important developmental molecules. Clinical aspects of development are covered throughout in boxed sections of text. First-rate illustrations complete this essential package. Integrates contemporary developmental knowledge with classical embryological understanding.
Interprets complex molecular developments, to help you learn how exactly the embryo develops. Presents first-rate clinical photos and clear drawings, to help you to memorize and understand normal and abnormal development. Uses clear sections within the chapter and summaries at the end of each to help you navigate this complex subject. Includes review questions at the end of each chapter to help you assess your knowledge.
This qualitative specificity of adhesion systems provides a mechanism for the assembly of different types of cell aggregate in close proximity, and also prevents individual cells wandering off into neighboring domains. If cells with different adhesion systems are mixed they will sort out into separate zones, eventually forming a dumbbell-like configuration or even separating altogether Fig.
In addition to the qualitative aspect of specificity, it is also known that cell-sorting behavior can result simply from different strengths of adhesion of the same system. The process is based on the existence of small random movements of the cells in the aggregates, and the same final configuration will be reached from any starting configuration, for example from an intimate mixture of the two types, or from blocks of the two types pressed together.
Classification of morphogenetic processes Similar repertoires of morphogenetic processes are re-used repeatedly in different developmental contexts Fig. This type of analysis was used to investigate the posterior group mutants in Drosophila and to show that nanos was the last-acting member of the pathway see Chapter If members of a set of mutants have one of two opposite phenotypes then the genes may again code for the successive steps in a pathway but it is likely that some or all the steps will be repressive events rather than activations.
Figure 3. Pigment is made in the spots of segment 2 following the operation of a pathway of three genes in which a represses b which represses c which represses pigment formation.
Normally gene a is active everywhere except the spots, so only the spots become pigmented. It is possible to deduce the sequence of action of the genes by examining the phenotype of the double mutants.
In each case the phenotype of the double mutant is the same as that produced by the mutant of the later acting of the two genes. By looking at the phenotype of each double mutant combination, the genes can be arranged into a Pigmented Pigmented Fig. Normally, a gene a is inactivated in three spots of segment 2, resulting in the formation of dark pigment. In a loss of function mutant of a or c the whole organism is pigmented.
In a loss of function mutant of b there is no pigment. The phenotypes of the double mutants show that b must act after a and before c. Among many other examples, this type of analysis was used to order the dorsal group genes in Drosophila see Chapter Repressive pathways are remarkably common and they can cause much confusion. The best way to understand them is, as in Fig. In developmental biology the two conditions often refer two regions within the embryo where the same pathway is under different regulation, for example the dorsal and ventral sides.
These methods are examples of epistasis analysis, because if one gene prevents the expression of another it is said to be epistatic to it. Another method of ordering gene action in development depends on the use of temperature-sensitive mutations. In contrast to the pathway situations, this does not depend on any particular relationship between different gene products and can be used to order events in time which are mechanistically quite independent of each other.
Temperature-sensitive mutants are those which display the phenotype at a nonpermissive usually high temperature, and do not show a phenotype at the permissive usually low temperature. They are frequently weak loss-of-function alleles. They arise from changes in the conformation of the protein product which are sensitive to changes in temperature in the range compatible with embryonic survival. The time of action of a gene may be deduced by subjecting groups of temperature-sensitive mutant embryos to the nonpermissive temperature at different stages of development.
If the Developmental genetics organism ultimately displays the mutant phenotype, this means that the gene was inactivated at the time of its normal function, in other words that the gene was required during the period of the high temperature exposure. An example would be the time of action of the gene cyclops, which is needed for the induction of the floor plate in zebrafish. Temperature-sensitive mutants are more use in poikilothermic organisms such as C.
Genetic mosaics It is sometimes possible to make organisms that consist of mixtures of cells of different genotypes. These are called genetic mosaics and can be useful as they provide information about where in the embryo a particular gene is required.
For instance an embryo may consist of two territories, A and B, and a particular mutant shows a defect in B. We can consider two informative types of genetic mosaic Fig. One has prospective A cells mutant and prospective B cells wild type, while the other has a u 25 prospective B cells mutant and prospective A cells wild type.
If the organism with B cells mutant shows the abnormal phenotype, then we say that the mutant is autonomous; it affects just the region in which the gene is normally active. However, if the organism with A cells mutant shows the abnormal phenotype, then the mutant is nonautonomous because it is affecting a structure outside the domain of action of the gene. Nonautonomy means that there must be an inductive signaling step that is affected by the mutation.
However, it does not necessarily mean that the mutant gene itself codes for a signaling factor, as failure of the signaling event can be a downstream consequence of the mutation. Genetic mosaics have been widely used in Drosophila.
A very useful type is made by pole-cell transplantation and consists of germ cells of one genotype in a host of a different genotype. Such mosaics have enabled the understanding of factors controlling the patterning of the oocyte as a result of interactions with the somatically derived follicle cells of the egg chamber see Chapter Mosaics have also been used in C. In zebrafish, mosaics can be created by grafting as there is quite a lot of early cell mixing to disperse the labeled cells.
In mammals, the term mosaic is usually reserved for a naturally occurring organism composed of two genetically dissimilar cells e. X inactivation mosaic, see Chapter 10 and the term chimera is used for embryos made experimentally by cell injection or aggregation of blastocysts.
Genetic mosaics should not be confused with embryos said to show mosaic behavior see Chapter 4. This means that surgical removal of parts causes a defect in the final anatomy corresponding exactly to the fate map. Mosaic behavior is contrasted to regulative behavior and has nothing to do with genetic mosaics. Screening for mutants b c d e Fig. In b and c genetic mosaics are made in which the red tissue is null mutant and the green tissue is wild type. In b spots appear in the wild-type zone so the gene must have an utonomous function corresponding, for example, to the wild-type expression pattern in d.
In c a spot appears in the zone of mutant tissue so the gene must have a nonautonomous function, corresponding, for example, to the expression pattern in e. The term forward genetics is sometimes used to describe investigations that start with the discovery of an interesting mutant phenotype. Reverse genetics, by contrast, refers to functional investigations on a known gene. Many interesting mutants have arisen spontaneously, but even more have been recovered in large-scale screens on Drosophila, C.
The details of these screens can be very complex, particularly for Drosophila in which there are many selective tricks for reducing the total number of individuals to be dealt with. But the principle is simple and relies just on basic Mendelian genetics. The following description approximates to the procedures used in zebrafish screens, although is still slightly simplified Fig.
A group of males will be mutagenized, for example by treatment with a chemical mutagen. This illustrates the simplest possible type of screen for zygotic recessives.
Each F1 individual is outcrossed to generate a family at the F2 generation. The mutagenized males are mated to normal females, producing an F1 offspring generation. Each of the F1 individuals is likely to carry a mutation in heterozygous form, and each is likely to carry a different mutation from all the others. So each F1 individual is put in a separate container for further mating to a wild-type animal.
This produces a family at the F2 generation. If the F1 individual did carry a mutation, then half the F2 individuals will be heterozygous for it. A set of test matings is carried out between pairs of individuals within each F2 family. The F3 generation is examined and scored at the embryo stage. By definition developmental mutations are those which perturb the anatomy of the organism, so the homozygous mutants should be visibly abnormal. They may be detected simply by examination of the embryos under the dissecting microscope, or if the screen is more focused, following immunostaining or in situ hybridization to display a particular structure or cell type under investigation.
Any mutation that disables a gene essential for early development is quite likely to be lethal and prevent development after the time of normal gene function. So the homozygous mutant F3 embryos may well die at an early stage, and they need to be examined early on before they degenerate.
The overall screening procedure has various elements of randomness. It is quite likely that F2 families will contain no mutation, or none giving an abnormal phenotype, in which case they are discarded. It is also possible for an F3 phenotype to be caused by more than one mutation in the original sperm. If there are two mutations showing independent segregation then the F3 generation will actually be a 9 : 3 : 3 : 1 mix of normal, single homozygotes of each type and double homozygotes.
This may not be apparent immediately, but can be resolved by further breeding. In Drosophila there are some very sophisticated methods for reducing the labor involved in screens. The most important is the use of balancer chromosomes. These have multiple inversions which mean that there is no recombination between the balancer chromosome and its wild-type homolog. They also carry a recessive lethal mutation, so flies with homozygous balancers are not viable.
They also carry some marker gene that will enable all flies carrying the balancer in one copy to be easily identified. The uses of balancers are numerous, but one of the most important is in the simple maintenance of a recessive lethal mutant line.
This is shown in Fig. The line carries one copy of the balancer chromosome and one copy of the homologous chromosome bearing the mutation. In each generation a 1 : 2 : 1 ratio is produced of homozygous balancer, heterozygous and homozygous mutant. The heterozygotes are the only viable offspring and serve to maintain the line. The homozygous mutant embryos are available for experiments.
This means that a line can be maintained by repeated mating with no need to test individuals to see whether they are heterozygotes. Cloning of genes Developmental genetics existed long before molecular cloning was introduced, but it is now regarded as essential to clone any u 27 gene of interest identified by mutagenesis. This caused considerable difficulty in the past but is much easier today with the availability of high-resolution genome maps and genome sequences.
A gene is regarded as cloned when the complete coding sequence is incorporated into a bacterial plasmid, or other cloning vector, so that it can be amplified and purified in a quantity suitable for use in any of the types of investigation now described as reverse genetics.
Most of the developmentally important genes in Drosophila were cloned by inducing mutations with a transposable element, the P-element. Once it had been shown that a P-element had integrated into the locus of interest then it could be used as a probe to isolate DNA clones from a genomic library. Nowadays, in most cases for experimental organisms as well as for human genetics, genes are cloned by positional cloning. Here a mutation is mapped to very high resolution using microsatellite polymorphisms or restriction fragment length polymorphisms.
There are many of these scattered through the genome and so long as the genome has been sequenced all their positions should be known. So long as enough offspring can be produced it is now possible to map a mutation to the specific locus using a single cross.
The procedure is shown in principle in Fig. It is crossed to an individual of another strain in which most of the polymorphic loci are different. These are then all individually typed for a selection of the polymorphic markers.
As shown in Fig. It will be necessary to go through several cycles of mapping, using the same DNA samples with markers that are more and more closely spaced around the mutant locus. Eventually a small chromosome region will be identified which is known from the genome sequence only to contain a few genes. Each of these is then evaluated as a candidate.
One consideration is the putative nature of the protein deduced from the sequence, for example if the mutation has a cell autonomous action it is unlikely to code for a signaling molecule. Another is the expression pattern of the candidate gene relative to the domain of action of the mutation. A gene that is not expressed in the relevant region is unlikely to be a good candidate.
The expression pattern can be established using in situ hybridization see Chapter 5 with probes designed from the known sequence. Once a good candidate has been found the mutant DNA can be sequenced at that locus to see if it does, in fact, contain a mutation.
Genome sequencing has finally enabled reasonably accurate estimates to be made of gene numbers, and hence indirectly of the complexity of living organisms. In terms of protein-coding genes, free-living bacteria have about — genes; unicellular eukaryotes like yeast about ; invertebrate animals like Drosophila and C.
The number for vertebrates is considerably lower than previous estimates but it should be remembered that complexity arises not only from the number of genes but also from the number of distinct proteins which may be produced by alternative splicing and post-translational modifications. It is Fig.
This is a very simplified presentation of the principle of positional cloning. The mutation to be cloned is in a gene called g. Three markers are considered, of which the parental strains have alleles A,B,C or a,b,c. Mutant or wildtype F2 individuals are analyzed using PCR primers for each of the polymorphic loci. The reaction mixtures are separated by gel electrophoresis and each specific allele is indicated by a DNA fragment of a particular size.
This indicates that the mutant locus lies near to the Aa locus. Transgenesis The scope of genetics has been considerably expanded in the molecular era such that many of the genetic variants used in experiments are not produced by mutagenesis, but by more sophisticated and directed methods. Such lines of animals are often called transgenic in popular or legal parlance. In biology it is more usual to reserve the terms transgenic and transgenesis for cases where an extra gene has been introduced into the Developmental genetics genome, as opposed to knockouts in which a gene has been removed.
A transgene is simply a gene that has been introduced into an organism by transgenesis. Methods for transgenesis exist for mouse, Xenopus, zebrafish, Drosophila, and C. In the mouse and zebrafish, the gene is introduced by injecting DNA into the fertilized egg. All methods of transgenesis share the property that the integration site in the genome is random, or at least not controlled.
Transgenes are usually designed so that their expression is regulated by a promoter within the insert and they are as far as possible immune from the effects of the position within the genome at which they have integrated. However, they are sometimes designed specifically to probe the local environment. An enhancer trap consists of a reporter gene such as lacZ see Chapter 5 coupled to a minimal promoter that can bind the transcription complex but has very low activity.
Enhancer trap lines have found considerable use, particularly in Drosophila see Chapter The gene trap is a related method for identifying functional genes by integration and has been used a lot in the mouse see Chapter There is no satisfactory method of transgenesis for the chick, but considerable use has been made of retroviruses to introduce genes into embryos by infection.
Retroviruses are RNA viruses and, after infection of a cell, the genome is reverse transcribed to DNA which becomes integrated in the host genome.
If an additional gene is inserted into the viral genome this too will be integrated and expressed under the control of one of the viral promoters. Infected cells make and export virus particles which infect the neighboring cells, and therefore in a few days the infection, and the transgene, spreads through the embryo. As this may eventually have harmful consequences for the embryo, the experiments are usually timed so that just the region of interest has been infected at the critical time.
Note that the type of virus used for genetic modification is replication competent, while those used for clonal cell labelling see Chapter 4 are replication incompetent. Targeted mutagenesis The introduction of specifically engineered mutations at a desired site in the genome is a technology that has been brought to a high level in the mouse, but is not generally available for other species. It has mainly been used to produce knockouts, or loss-of-function mutations in particular genes selected because they were thought likely to be of developmental or medical importance.
Targeted mutagenesis depends on homologous recombination, which is the direct replacement of a gene by a u 29 modified version made in vitro. If DNA is added to cells or embryos, most of the integration events will occur in the wrong place and so there needs to be a selection step to isolate the homologous recombinants that have replaced the endogenous gene.
This has to be done on tissue-culture cells, so the applicability of targeted mutagenesis depends on the availability of cells that can be grown in culture and can then be reintroduced into an embryo. Because tissue culture is not well developed outside the mammals this effectively limits targeted mutagenesis to mammals.
In the mouse there are embryonic stem cells that can be engineered and then reincorporated into embryos to produce germ-line chimeras from which the new strain can be bred see Chapter In several species of domestic mammals it has proved possible to clone whole animals by nuclear transplantation from primary cultures of fetal cells into enucleated oocytes see Chapter 2.
This should make it possible to develop targeted mutagenesis also for these species as the necessary selective steps can be done in tissue culture. Other ways to inhibit gene activity For the species in which targeted mutagenesis is not possible, other methods for producing specific inhibition of known genes have been developed. They are inherently less specific than mutation and when using them it is important to ensure that experimental tests of specificity have been carried out.
One method involves the design of dominant negative reagents. For example a transcription factor lacking its DNA binding domain may often act as a dominant negative because when overexpressed it will sequester all the normal cofactors needed by the wild-type factor, or it may form inactive dimers with the wild-type factor.
It does not need to be a homologous recombination. Alternatively the mRNA for the dominant negative protein can be produced in vitro and injected into the fertilized egg. This is a technique that is suitable for organisms with large eggs, like Xenopus and the zebrafish, because they are easy to inject and the injected mRNA can exert its effect on early developmental events before it is degraded or diluted by growth.
Secondly, there is the domain swap method, used extensively for transcription factors. Because transcription factors have a modular design see Appendix it is possible to replace an activating region with a repressing domain or vice versa. The domain-swapped factor will still bind to the same site in the DNA but instead of activating its target genes it will repress them or vice versa.
This is not quite the same as a loss-of-function mutation, since there will be an active repression of any gene to which the target factor binds, and these genes would not necessarily be inactive following a simple ablation of the transcription factor.
Again this can be introduced either by transgenesis or by injection of mRNA into the fertilized egg. The usual inhibitory 30 u Chapter 3 domain used in this type of experiment is that from the Drosophila gene engrailed, and the normal activating domain is that from herpesvirus gene VP The third strategy involves the use of antisense reagents.
When introduced into the embryo this will form hybrids with the normal mRNA, which are inactive as translation substrates and are often rapidly degraded.
There have been fashions for introduction of full length antisense mRNA, and for the external application of antisense oligodeoxynucleotides, but the currently favored methods fall into two groups: the use of morpholinos and the use of RNA interference RNAi. Morpholinos are analogs of oligonucleotides, in which the sugar-phosphate backbone is replaced by one incorporating morpholine rings. Unlike normal oligonucleotides they are resistant to degradation by the nucleases that are present in all cells and extracellular fluids, but because the usual four bases are linked to the resistant backbone with the correct spacing, they can still undergo hybridization with normal nucleic acids.
Morpholinos are usually synthesized to be about 20 residues long and are designed to be complementary to a region of the mRNA likely to be accessible, such as the translation start region.
The hybrid of morpholino and mRNA is not degraded but remains inactive for protein synthesis. Because there is no degradation of the mRNA it is necessary to show that specific protein synthesis has been blocked, which requires the availability of an antibody to the protein that can be used for Western blotting or immunoprecipitation or in situ immunostaining.
Morpholinos cannot generally penetrate cell membranes and so their main application has been in early embryos of free-living embryos where they can easily be administered by intracellular injection, namely Xenopus, zebrafish, sea urchins, or ascidians.
These enter a silencing complex that can bind to, unwind, and cleave mRNAs that contain complementary sequence. In mammalian cells the long dsRNA causes nonspecific inhibition of translation, but the short 21—23 bp length processed fragments do not, and can be used directly to bring about destruction of specific mRNAs. Because it is possible to make large libraries of dsRNA this method is now being used instead of chemical mutagenesis to conduct screens. It is particularly suitable for C.
A final method of specific inhibition is treatment of the embryo with a specific neutralizing antibody directed against the protein product of the gene of interest. Antibodies will not penetrate intact embryos and so they must be injected at the site of interest.
A common problem with this method is that most antibodies that bind to a particular protein will not neutralize its activity, so it is necessary to have some independent test to show that the antibody does, in fact, neutralize the target protein.
Some of these inhibitory techniques can be used in transgenic mode, but they are very often used as transient, nongenetic procedures. As mentioned above, it is easy to inject substances into Xenopus or zebrafish embryos, and it is also easy to treat later organ cultures from mammalian or chick embryos. This can be very useful so long as the inhibitor is able to penetrate to the site of action.
Gene duplication Gene duplication is probably the major source of evolutionary novelty. If a gene becomes duplicated then the constraints on changes to its sequence become relaxed.
At one extreme, one copy could continue to be the functional gene and the other copy could accumulate mutations such that it acquired a novel and advantageous function. Alternatively the second copy could accumulate deleterious mutations until it became nonfunctional, and maybe eventually not even expressed a pseudogene. More usually, both copies will accumulate some sequence divergence such that they carry out subsets of the original function.
Soon after the duplication the overlap in function will be considerable, while after millions of years the functions will diverge. For example the cyclops and squint genes of the zebrafish arise from duplication of the nodal gene which encodes a critically important mesoderm-inducing factor in vertebrate development, but they have diverged in function such that they act at different developmental stages see Chapter 8. The extreme case of gene duplication occurs when the entire genome becomes duplicated, with a doubling of chromosome number.
This is called tetraploidization, as the resulting organisms are tetraploid instead of diploid. Tetraploidization can produce a vast array of new genes instantaneously and so enormously enlarge the adaptive possibilities for the line of descent. The pattern of multigene families in vertebrates suggests that two tetraploidization events may have occurred at the time of the origin of vertebrates, temporarily boosting their gene number from about 20, to about 80, This may account for their subsequent adaptive radiation and evolutionary success, although the count of protein-coding genes in extant vertebrates suggests that the number has been much reduced in subsequent evolutionary time back to about 30, It also seems that further tetraploidizations have occurred in various lineages.
These are known as pseudoalleles. They look like alleles, and generally have the similar expression patterns and functions, but they are not alleles because they occupy distinct genetic loci.
Overall, the book is concise, well structured, and illustrated. I can highly recommend it. This effort is no exception.
0コメント