Iiag Notes / Developmental Biology R.M. TwymanCONTENTS Abbreviations Preface Section A ~ First principles Al Ad AB Aa AS AG Ay Basic concepts in development biology Cell fate and commitment Mechanisms of developmental commitment Mosaic and regulative development Maintenance of differentiation Pattern formation and compartments Morphogenesis Section B - Experimental developmental biology BL B2 BS B4 Model organisms Developmental mutants ‘Transgenic organisms in development Cellular and microsurgical techniques Section C - Genes in development ca Q a ca co Gene expression and regulation Chromatin and DNA methylation Signal transduction in development The cell division cycle The cytoskeleton, cell adhesion and the extracellular matrix Section D ~ Unicellular models DI bz DB Sporulation in Bacillus subtilis Mating type switching in yeast Aggregation and culmination in Dictyosteliuan discoideum Section E - Sex, gametes and fertilization El E2 E3 Ea ES Germ line specification Germ-cell migration Gametogenesis Gamete recognition, contact and fertilization Sex determination Section F - Cleavage and gastrulation FL F2 FB Cleavage Gastrulation in invertebrate embryos Gastrulation in vertebrate embryos Section G - Single cell specification GI G2 B G4 Early animal development by single cell specification Cell specification and patterning in Caenorhabditis elegans Ascidian development Early development of molluscs and annelids vii ix 45 45 49 56 64 69 69 7 85 95 100 107 107 12 17 125 125 130 135 145, 151 161 161 172 77 187 187 190 197 201vi Contents Section H ~ Axis specification and patterning in Drosophila H1 Molecular aspects of embryonic pattern formation in Drosophila H2_ Anteroposterior axis specification in Drosophila H3 Gap genes Hd Molecular control of segmentation H5 Homeotic selector genes and regional specification H6 —Dorsoventral axis specification and patterning Section I - Axis specification in vertebrates Tl Early patterning in vertebrates 12 The vertebrate organizer 13 Leftright asymmetry in vertebrates Section J ~ Fate of the ectoderm Jl The ectoderm: neural induction and the epidermis J2_ Patterning the anteroposterior neuraxis J3._ Patterning the dorsoventral neuraxis J4 Shaping the neural tube JS Neurogenesis, Jé The neural crest J7 Neuronal connections Section K —- Mesoderm and endoderm K1_ Mesoderm induction and patterning K2_ Somitogenesis and patterning K3_ Somite differentiation Ka Mammalian kidney development K5 Heart development K6 —Endoderm development Section L - Organogenesis in invertebrate model systems Li Vulval specification in C. elegans 2 Pattern formation in imaginal discs L3 Drosophila eye development Section M — Vertebrate limb development M1 Initiation and maintenance of limb growth M2 Patterning and morphogenesis in limb development M3 Limb regeneration Section N - Plant development N1 Plant vs. animal development N2_ Development of the plant embryo N3__Development of the seedling N4 Shoot and root meristems N5 Leaf development N6 Flower development Further reading, Glossary of acronyms Index 205 205 21 219 25 235 244 251 251 257 263 269 269 275 287 292 296 302 312 323 323 330 337 341 347 350 357 397 361 367 371 371 381 390 397 397 405 410 a4 423 429 439 445 447ABBREVIATIONS 3-UTR ABA ADMP AEC AER ALCS ANT-C AVE BDNF BMP BWS BX CAM CDK CNS DAG DIF ECM EDTA EGF EMS ENU FGF FLIP FSH GA GAP GDNF GEF GPCR GSk3 HOM-C IM IRAP 3’ untranslated region abscisic acid antidorsalizing morphogenetic protein apical ectodermal cap apical ectodermal ridge anterior-like cells Antennapedia complex anterior visceral endoderm brain-derived neurotrophic factor bone morphogenetic protein Beckwith-Weidemann syndrome Bithorax complex cell adhesion molecule cyclin dependent kinase central nervous system diacylglycerol differentiation inducing factor extracellular matrix ethylene diamine tetra-acetic acid epidermal growth factor ethylmethane sulfonate N-ethyl-N-nitrosourea fibroblast growth factor floral initiation program follicle stimulating hormone gibberellic acid GTPase activating protein glial-derived neurotrophic factor guanine nucleotide exchange factor G-protein-coupled receptor glycogen synthase kinase-3 homeotic complex inner cell mass intermediate filament-asso protein IP, JAK JNK LGN LH LRD MAP MAR MBT MTOC N-CAM NGF NIC PGC PKA PKC PMc PMZ PSE RAGE RAM REMI SAM SMC STAT TCR Ti plasmid TIFs uv PCs Xa inositol-l 4,5-trisphosphate Janus kinase Jun N-terminal kinase lateral geniculate nucleus leutenizing hormone left-right dynein microtubule associated protein matrix attachment region midblastula transition microtubule organizing center neural cell adhesion molecule nerve growth factor node-inducing center primordial germ cell protein kinase A protein kinase C primary mesenchyme cell posterior marginal zone prestarvation factor recombinant activation of gene expression root apical meristem restriction enzyme-mediated integration shoot apical meristem secondary mesenchyme cell signal transducer and activator of transcription T-cell receptor tumor-inducing plasmid transcriptional initiation factors ultraviolet vulval precursor cells active X inactive X X-inactive-specific transcript zone of polarizing activityPREFACE Developmental Biology now unites all the biological sciences, and is assuming more and more impor- tance in fields such as medicine and agriculture. For this reason, many more students are required to learn about development as a short module forming part of a larger course. Conscious of the extra pressure this places on students and their teachers, I have tried to write a book that provides a clear summary of the principles of development in a concise and easily manageable format. Development has always been perceived as a difficult subject, and this reflects its origins in the study of diverse organ- isms using different approaches: simple observation, experimental embryology, genetics and molecular biology. Over the last 10 years, these disparate elements have come together resulting in a new era for developmental biology, which is gene-based and in which the complex cell behaviors that guide development are being dissected at the molecular level. This has shown that fundamentally similar mechanisms guide the development of all organisms, a theme that is emphasized throughout the book. This book focuses largely on the principles of embryonic development, while coverage of post- embryonic development is limited, except in plants. There is also no attempt to describe development in evolutionary terms. This slight reduction in coverage has been necessary to keep the book to an acceptable length and price, but it should still meet most of your course requirements, This book would not have been possible without the valuable help and advice of many friends and colleagues. In particular [ would like to thank those who took the time to read and comment on indi- vidual topics and sections: Gavin Craig, Dylan Sweetman, Phil Gardner, Jane Pritchard, Christine Holt, Victoria James, Manuela Costa, Mark Leech, Eva Stiger, Ajay Kohli, James Drummond, Roz Friday, Derek Gatherer, and especially Clare Hudson. I would also like to thank my many past and present students at Gonville and Caius, Newnham, New Hall, Girton, Petethouse, and King’s Colleges in Cambridge, who acted as guinea pigs for various parts of the book and helped to shape its content and presentation. I would like to thank my teachers, who cultivated my interest in development: Helen Baker, Trevor Jowett, Liz Jones, David Stott, and Bob Old. would also like to acknowledge Bob Old (University of Warwick), Nigel Unwin (MRC Laboratory of Molecular Biology, Cambridge) and espe- cially Paul Christou (Molecular Biotechnology Unit, John Innes Centre, Norwich) for their support and ‘encouragement at different stages of the project. Finally of course, thanks to the dedicated team at BIOS Gonathan Ray, Victoria Oddie, Lisa Mansell, Ailsa Henderson, Andrea Bosher, and Rachel Offord) and for the advice and encouragement of David Hames Cover images. Left Drosophila larval neuromuscular junction courtesy of James Drummond. Right: Ascidian larva showing localization of Otx mRNA courtesy of Clare Hudson, Upper inset panel: Section of mouse brain showing hippocalcin gene expression courtesy of Bill Wisden and Alison Jones. Lower panels: Arabidopsis thaliana wild type flower (left), apetala2 mutant (middle) and apetala2 /agamous double ‘mutant (right) courtesy of Robert SoblowskiTo my parents, Peter and Irene, and to my children, Emily and LucySection A - First principles A1 BASIC CONCEPTS IN DEVELOPMENTAL BIOLOGY Key Notes Development’ 4 Development is the process by which a complex multicellular organism arises from a single cell. It involves an increase in cell number, differentiation, pattern formation and morphogenesis, as well as net growth. Development is a gradual process, so the complexity of the embryo increases progressively. Although animals show great diversity, the development of most animals proceeds through a number of common stages. These include fertilization, cleavage to form a group of blastomeres, gastrulation to reorganize the structure of the embryo and generate the three germ layers, neurulation (formation of the nervous system) and organogenesis (the development of individual organs), Flowering plants undergo double fertilization to produce the zygote and the endosperm tissue that nourishes the embryo. Embryonic development in plants comprises a series of stereotyped cell divisions to generate a mature embryo comprising three fundamental cell layers and containing regions carresponding to the shoot, root and cotyledons of the seedling. After germination, all parts of the mature plant are produced from two small populations of proliferative cells established in the embryo, the shoot and root meristems. Although development involves both cell division and growth, these processes can occur independently. During the cleavage divisions that occur in early animal development, there is an increase in cell number without growth, so that the egg is divided into a series of progressively smaller cells. Later in development, cell division and growth occur together, although growth may occur without cell division through changes in cell size and the deposition of materials such as bone into the extracellular matrix. Differentiation is the process by which different cell types are generated, Cells become structurally and functionally specialized by synthesizing different proteins. Differentiation thus reflects the activation and maintenance of different patterns of gene expression. All embryos of a given species have a similar structure or body plan because the cells become organized in the same way. The body plan is progressively filled with detail as development proceeds. To organize themselves in this way, cells must know where they are in relation to other cells in the embryo. This is achieved by giving each cell a positional value in relation to the principle embryonic axes, often in response to a morphogen gradient. Later in development, pattern formation generates: Section A ~ First principles Genes in development Related topics the fine details of emerging organs, A variety of different mechanisms involved, some intrinsic and others requiring cell-cell interactions. Morphogenesis is the creation of shapes and structures. This is governed by different forms of cell behavior, including differential rates of cell proliferation, changes in cell shape and size, the movement of cells relative to each other, cell fusion and cell death. Alll the major processes necessary for development are dependent, either directly or indirectly, on proteins. Genes therefore control development by determining where and when proteins are synthesized in the embryo, and thus how different cells behave. The analysis of developmental mutants has shown that many genes with important roles in development encode transcription factors and components of signaling. pathways. Mechanisms of developmental Morphogenesis (A7) commitment (A3) Gene expression and regulation Maintenance of differentiation (A5) (C1) Pattern formation and Plant vs animal development (NI) | compartments (A6) Development Overview of animal development Development is the process by which a multicellular organism arises, initially from a single cell. Development is progressive, i.e. a simple embryo, comprising few cell types organized in a crude pattern, is gradually refined to generate a complex organism with many cell types showing highly detailed organization. This gradual developmental strategy is known as epigenesis, in contrast to the earlier theory of preformation, which suggested that the early embryo comprised a miniature version of the adult, and development consisted entirely of growth. In fact, development involves five major overlapping processes: growth, cell division, differentiation, pattern formation, and morphogenesis (Fig. 1). Early development is usually studied in the context of the embryo as a whole. Later, developmental biologists concentrate on particular model developmental systems, which may be used to illustrate specific principles. For example, mammalian kidney development is used as a model for reciprocal cell-cell inter- actions (Topic K4), while vertebrate limb development is a useful model for pattern formation (Section M). Much of this book is devoted to animal development, so this first Topic provides an opportunity to summarize some of the developmental stages common among animals and introduce some key terms. Animals show great diversity, but early development in most animals involves a common series of events (Fig. 2). Development usually begins with fertilization, when a sperm makes contact with the egg and the male and female nuclei fuse generating a diploid zygote opic E4). Fertilization is followed by cleavage, a series of rapid and synchro- nous cell divisions that occurs in the absence of growth and divides the large egg into a number of smaller cells, termed blastomeres (Topic F1). Uniquely in insects, the zygotic nucleus initially divides a number of times without cell division, so a multinucleate syncytium is generated. The blastomeres form when the nuclei migrate towards the periphery of the embryo and becomeAt - Basic concepts in developmental biology 3 Overview of plant development <>) T. Growth Cell Differentiation Pattern Morphogenesis division jormation Increase Increasein Diversification Organization Generation of in celinumber _of cell types. shapes and size structures Ci Gametes Fig. 1. An overview of developmental processes. enclosed by newly formed cell membranes. In all animal embryos, the result of cleavage is the formation of a ball or disc of blastomeres, often enclosing a fluid- filled cavity. Different terms are used to describe the embryo at this stage, depending on the species (e.g. blastula, blastodisc, blastocyst, cellular blasto- derm), The next stage of development is gastrulation, which involves a complex series of cell movements that reorganizes the embryo into three fundamental cell layers (Topics F2 and F3). These are called the germ layers (ectoderm, meso- derm and endoderm) and are present in all animal embryos.' Gastrulation is followed by neurulation, the development of the central nervous system (Section J). Once these fundamental processes have occurred, individual organs begin to develop according to their own programs (organogenesis). Plant and animal development are discussed separately in this book because there are some important and fundamental differences between plants and animals in terms of their developmental biology (Topic N1). However, itis also "Most animals are triploblastic, meaning that the embryo produces three germ layers, Some primitive animals (including coelenterates such as Hydra) are diploblastic, lacking the mesoderm layer. In these animals, a jelly-like secretion called the mesogloea separates the ectoderm and endocerm.| Fertiization | Eattycell | Formation of three Emergence of Post-embryonic : divisions —_|_ fundamental cell types body plan development i ne —— ne | oe os coe se Neurulation, Sood Gastrulation = soma v= CE>— Coa Ce _/ saica i Growth | i (Metamorphosis) ie Syncytium ! fi ! i | | i Pollen grain | i i | | i Stigma: Style: stereotypical Stereotypical I ro Zygor _ sions divisions @ Oo 7 1 — . L- Suspensor | Owe: ; Seeding Adult (from Globular Meristers (from embryo) meristems) Diploid central embryo _ i Fig. 2. Comparative summaries of the development of animals and flowering plants. sajdioupd ysaty ~ y wonseg,AM - Basic concepts in developmental biclogy 5 Growth and cell division Differentiation useful to consider some similarities between animals and plants in early development (Fig. 2). The summary provided here refers to flowering plants, Development begins with double fertilization. The male gametophyte (pollen grain) produces two sperm cells and a vegetative cell. When a pollen grain adheres to the stigma of a flower, the vegetative cell elaborates into a pollen tube, which grows down through the style and penetrates one of the ovules, The two sperm cells migrate down the tube into the female gametophyte (the embryo sac) which is inside the ovule. One sperm cell fuses with the egg cell to generate a diploid zygote, while the other fuses to the diploid central cell to generate the triploid endosperm cell. The endosperm layer of the seed is derived from this cell, and nourishes the growing embryo. Fertilization is followed by a series of stereotyped cell divisions to generate a ball of cells called a proembryo, attached to the ovule by a suspensor (Topic N2). There is no gastrulation-like stage in plants because relative cell movement is prevented by the rigid cell walls, Further growth and cell division produces an embryo organized radially into three principal cell layers, L1, L2 and L3, and organized axially into a series of organ-forming regions that correspond to the future cotyledons (storage organs) shoot and root of the seedling, and the proliferative shoot and root meristem populations that give rise to all adult structures. The function of the plant embryo is to produce the seedling, which emerges from the seed under environmental conditions suitable for germination, pushing a shoot towards the surface of the soil and the root downwards (Topic N3). All the organs of the adult plant (stem, leaves, flowers, roots) arise from the shoot and Toot meristems during post-embryonic development (Topic N4). Development usually begins with a single cell, the fertilized egg, and generates an organism with many cells. Although the egg is a comparatively large cell, it is still smaller than the mature organism that develops from it, Development therefore involves both growth and an increase in cell number. In very simple organisms, such as the nematode Caertorhabditis elegans, the cell number in the adult is less than 2000. Conversely, large animals such as humans comprise many trillions of cells. An increase in cell number is necessary to allow cells to specialize, organize into patterns and carry out different functions. In the earliest stages of animal development, the cell number increases in the absence of growth. During these cleavage divisions, the large egg is divided into a number of smaller cells as the cell cycle alternates rapidly between DNA replication and mitosis without any interstitial gap phases. The nature of these divisions is often instrumental in setting up the fundamental body plan of the embryo (see Topic F1). Later in development, growth can occur in the absence of cell division, as growth can be achieved not only by increasing cell number, but, also by increasing cell volume, changing cell shape or by the growth of extra- cellular structures generated by materials secreted from cells (e.g. cartilage and bone). Cell growth and cell division do not therefore contribute solely to the of the developing organism, but also to its shape and pattern. ize Differentiation is the process by which cells become structurally and function- ally specialized, allowing the formation of distinct cell types (e.g. neurons, erythrocytes and keratinocytes in animals; xylem, phloem and parenchyma in plants). The structural and functional specificity of a cell depends on the proteins it synthesizes. For example, the ability of red blood cells to carry oxygen reflects the synthesis of hemoglobin specifically in these cells. With verySection A - First principles Pattern formation Morphogenesis few exceptions, all cells of a given organism contain the same genetic informa- tion; thus differentiated cells are genetically identical but express different genes. The process of differentiation therefore involves the control and mainte- nance of differential gene expression. Cells may begin to express different genes if they inherit different cytoplasmic components at cell division, or if they receive signals from other cells or the environment. Such interactions are consid. ered in more detail in Topic A3. The maintenance of differential gene expression is discussed in Topic A5. Pattern formation is the process by which cells become organized in the embryo, initially to form the basic rudiments of the body plan, and later to generate the fine structure of individual organs. For development to succeed, each cell must behave in a manner appropriate for its position in the embryo, so that the correct differentiated cell types arise in the correct places and cells of similar type form regionally-appropriate structures (such as bones of different shapes and sizes in different parts of the body). The process by which cells become specialized according to their position is called regional specification. ‘The first patterning event that takes place in the embryo is axis specification, the establishment of the principal body axes (anteroposterior, dorsoventral etc; see Topic A6). These axes may be pre-determined by the distribution of maternal gene products in the egg (as occurs in Drosophila development; Section H), or may require a physical cue from the environment to influence gene expression or protein activity in the embryo itself (e.g. the point of sperm entry in Xenopus; Topic 11). Once the principal axes are established, the position of cells along each axis must be specified. This is often achieved by dividing an axis into individual developmental compartments and allocating each a posi- tional value. Several experimental systems have shown that positional values may be assigned according to the position of a cell in a morphogen gradient. This is covered in more detail in Topic A6, and several well-characterized devel- opmental systems based on this morphogen model are discussed in this book (e.g. early Drosophila development, Section H; vertebrate limb development, Section M). A diversity of patterning mechanisms occurs later in development, including asymmetric cell division, specific localized interactions between cells, and the generation of regular spacing patterns by lateral inhibition. In each case, the cell pattern that emerges reflects a pre-existing pattern of gene expression in the developing organ. However, a number of developmental systems also show spontaneous pattern-forming ability when known patterning genes are mutated, and this may reflect an underlying reaction-diffusion mechanism (Topic A6) Morphogenesis means the creation of structure and form, and reflects the many different types of cell behavior that help to shape tissues and organs. Cells may change their size and shape, they may adhere or disperse, they may remain quiescent or divide, they may move relative to other cells, they may fuse together and they may even die in the process of sculpting a particular devel- oping organ. In later development, morphogenesis can be regarded as a response to the developmental program, ie. a differentiated cell aware of its position in the embryo will behave in such a manner as to generate a regional appropriate structure. Earlier in development, however, morphogenesis is instrumental in driving the developmental program, e.g. by bringing cells together to undergo inductive interactions. This is illustrated by the complexAM ~ Basic concepts in developmental biciogy 7 Genes in development morphogenetic movements of gastrulation (ection F). The cellular basis of morphogenesis is discussed in more detail in Topic A7, Alll the processes that are essential for development ~ cell division, growth, differentiation, pattern formation and morphogenesis — are mediated ultimately by proteins (acting either directly, or as enzymes to produce other molecules). Proteins are encoded by genes, so development is controlled to a large degree by gene expression. The pattern of gene expression in the embryo determines where, when and in what quantity particular proteins are made and therefore governs the properties of each cell (Topic C1). The analysis of developmental mutants (Topic B2) can provide clues as to the functions of particular genes. Many genes shown to have important roles in development encode two partic- ular types of protein, transcription factors and components of signaling path- ways. Transcription factors are important because they control gene expression, and therefore act as coordinators of developmental processes. Signaling path- ways are necessary for cells to perceive external signals, either from other cells or from the environment. We return to the subject of transcription factors and signaling pathways many times in the course of this book.Section A - First principles A2 CELL FATE AND COMMITMENT Key Notes | The developmental ‘hieraschy. Related topics The number of different cell types in the embryo increases as development proceeds, but new cell types arise from particular pre- existing cell types through a hierarchical series of decisions. In animals, but not plants, such decisions tend to be irreversible, so there is a progressive restriction of cell fate and potency during development. Celllaie ahd potency] Cell fate describes the range of cell types a particular cell can give rise to during normal development, while cell potency describes the range of cell types that a cell can give rise to in all possible environments. The fate of a cell is dependent on both its potency (which is an intrinsic quality) and its interactions with other cells in the embryo. (Fate maps A fate map is a map of an embryo showing which parts of the embryo give rise to particular adult tissues, Fate maps are generated by marking or labeling cells in the early embryo and following their progress through development. In species that undergo stereotyped cell divisions, fate maps are accurate. In most species, there is a certain degree of random cell division and mixing, so fate maps are probabilistic. In animals, cells become committed to certain fates in stages, first reversibly and then irreversibly. A naive cell may receive information in the form of cytoplasmic determinants or inductive signals that specifies its fate, but this fate can be altered by placing the cell in a different environment. A cell becomes determined when its fate is irreversible This coincides with the loss of competence to follow alternative developmental pathways. Mechanisms of developmental Early animal development by single commitment (A3) cell specification (G1) Mosaic and regulative Plant os animal development (N1) development (A4) Maintenance of differ: (A5) The developmental hierarchy As development proceeds, the number of cell types in the embryo increases. However, new cell types do not arise randomly, but through a hierarchical series of decisions involving specific pre-existing cell types. One of the earliest events in animal development is the division of the embryo into the three germ layers: ectoderm, mesoderm and endoderm. Each of the germ layers then gives rise to a specific range of differentiated cell types as shown in Fig. 1. Similarly, the plant embryo is divided into three fundamental cell layers, L1, L2 and L3. EachAZ ~ Cell fate and cammitment 9 Blastula | i T 1 Ectoderm, Mesoderm, Endoderm Neural Neural Dorsal \Ventrolateral plate crest mesoderm mesoderm, Notochord Somites Limb bud! Epidermis CNS PNS Dermis Muscle Cartilage aa Visceral Gut bone Kidney Heart Biood mesoderm epithelia Fig. 1. Simplified developmental hierarchy in vertebrates. Cell fate and potency of these also gives rise to a particular range of differentiated cell types as shown in Fig, 2. In animals, these developmental decisions are usually irreversible. In vertebrates for example, once a cell has differentiated as ectoderm, its fate is fixed. It will eventually form either neural or epidermal tissue or a derivative of the neural crest, but can never give rise to liver or kidney tissue, which are specific to the endoderm and mesoderm lineages, respectively. Development in animals therefore comprises a series of decisions that progressively and irre- versibly restrict cell fate. Conversely, plants show much greater plasticity in their developmental hierarchy, and cells originally in one of the layers can switch fates relatively easily. ‘The fate of a cell is all the different cell types its descendants can become during normal development. In this context, normal development means development undisturbed by experimental manipulation. A cell may differentiate in an abnormal way if it is placed in an unusual environment e.g. by grafting, so it is, exposed to other cells with which it normally does not come into contact. The term potency is used to describe the entire repertoire of cell types a particular cell can give rise to in all possible environments. The potency of a cell is an intrinsic property and is greater than or equal to its fate; the fate of a cell Proembryo Li (protoderm) L2 (ground meristem) L3 (procambium) Epidermis Subepidermis Cortex Vascular Pith tissue Fig. 2. Simplified developmental hierarchy in flowering plants.10 Section A - First principles Fate maps depends on its potency and its environment (e.g. its contact with other cells in the embryo). In animal development, cell fate and potency are progressively restricted. For example, at the 16-cell stage, each blastomere of a mouse embryo is totipotent; i.e. it can potentially give rise to every cell type in the adult if transferred to another embryo. Later, the morula differentiates to form the inner cell mass and the trophectoderm (Topic F1). The inner cell mass gives rise to the embryo, and differentiates to form the three germ layers as well as other extra-embryonic structures. At this point, individual cells are still pluripotent since they can generate several different cell types, but they are no longer totipotent, since certain fates are now unavailable. Cell fate becomes increasingly restricted until a cell is terminally differentiated (can form only a single cell type). Some cells, eg. hepatocytes, continue to divide but only produce identical daughters. Other cells, e.g. neurons, become quiescent (Le. they exit from the cell cycle and do not divide further). Stem cells are exceptional because they are never terminally differentiated, Instead of dividing to produce two identical daughters, stem cells produce dissimilar daughter cells, only one of which undergoes terminal differ- entiation (Fig. 3). The other daughter is a replacement stem cell, and in this way stem cells can continually replenish the organism with a given cell type. This is useful for cells that are depleted in the course of their normal function (e.g, skin cells, cells lining the gut and genitourinary lumena, and blood cells). © Hepatocyte © stem cel cm ro O O Hepatocyte Stem cell CQ (__) Keratinocyte rah ae © OO O Hepatocyte ee Stem cell ©) (__) Keratinocyte Fig, 3. Terminally differentiated cells compared to stem cells. In plants, cell fates are progressively restricted but differentiation does not correspond with terminal restriction in potency. Differentiated plant cells can change fates relatively easily if moved to a new position, and even fully differ- entiated plant cells can regenerate an entire new plant if placed in isolation. Therefore, while the fates of plant cells may be restricted during development, the cells remain totipotent even when they have differentiated. This is one of the fundamental differences between animal and plant development (Topic N1). Certain animal cells also show a limited developmental plasticity, which allows eg, limb regeneration in insects and amphibians (Topic M3). In these particular cases, certain differentiated cells remain pluripotent even when they are differ- entiated, A fate map is a map of an embryo or other developing system showing which adult tissues are formed from each region during normal development (Fig. 4). The term ‘presumptive’ is used to describe the fate of each region. For example, the part of the Xenopus blastula-stage embryo that will become the neural plateA2~ Cell fate and commitment "4 Levels of developmental commitment ‘Neural Kidneys, — Blood Mesenchyme Fig. 4. Fate map of the Xenopus blastula ~ lateral view. is called the presumptive neural plate. Fate maps can be generated by labeling particular cells and following their progress through development. This can be achieved by staining the cell surface with a vital dye, injecting a fluorescent dye into the cytoplasm, or using cells grafted from a different species that can be distinguished from those of the host (e.g. chick-quail chimeras). Alternatively, genetic markers can be used, e.g. reporter genes or mutations that generate pigmentation defects. This is especially useful where the tissue is not accessible, eg. plant meristems (Topic B4). Later, each labeled or marked cell will give rise toa clone of differentiated cells, confirming the fate of the original cell. In some systems, such as the Caenorhabditis elegans embryo and the devel- oping root of Arabidopsis thaliana, early fate maps are accurate because develop- ment involves a sequence of stereotyped cell divisions. In other organisms, there is a greater or lesser degree of random cell division and cell mixing, and fate maps are probabilistic, i.e. cells may have a choice of fates and likelihoods are attached to alternatives. Where cell mixing is totally random, as in the early zebrafish embryo, it is impossible to derive a fate map. In organisms such as C. elegans, where every cell division during development is stereotyped and each individual has the same number of cells, it is also possible to construct a diagram of cell lineage (this resembles a family tree and can trace every cell in the adult back to the egg). Such diagrams are useful, especially in the study of lineage mutants (Topic G1) but unlike fate maps, do not show the relative posi- tions of the cells in the embryo. Cell labeling can also show whether cells have been allocated to particular developmental compartments, a subject considered, in more detail in Topic A5. As cell fate becomes restricted following each decision in the developmental hierarchy, cells are said to become committed to a certain fate (or develop- mental pathway). In animals, commitment occurs in stages, initially reversible and then permanent (Fig. 5). In plants, commitment appears always to be reversible, so the following discussion relates only to animals. An uncommitted cell can be described as naive, meaning that it has received no instructions directing it along a particular developmental pathway. The fate of a cell is said to be specified if the cell is directed to follow a certain develop- mental pathway and does so when placed in isolation, which should provide a neutral environment. Specification may occur if a cell inherits a particular cyto- plasmic determinant or receives inductive signals from another cell. However, that same cell placed in a different environment, such as in contact with other cells, may be respecified by its interaction with those cells. This shows that the12 Section A - First principtes ee ==> Cytoplasmic Loss of competence Cell specific determinants for alternative fates, gene expression or or Induction _ Positive feedback loop or Chromatin remodeling Fig. 5. Stages of developmental commitment. commitment at this stage is reversible. The fate of a cell is said to be determined if it cannot be changed, regardless of the cell's environment. This shows that the commitment has now become irreversible. Determination to follow one developmental pathway coincides with loss of competence to follow alternative pathways. Cytoplasmic determinants, induc- tive signals and the concept of competence are discussed in more detail in Topic A3.Section A - First principles A3 MECHANISMS OF DEVELOPMENTAL COMMITMENT Key Notes ‘How cells differentiate Induction © | Tusiructive and permissive induction’ (/ atta hibition anu! the community To differentiate, cells must begin to synthesize new proteins. This requires the selective expression or activation of regulatory molecules such as transcription factors that control which proteins are synthesized in the cell. Such regulatory molecules could be inherited as cytoplasmic determinants or activated by external signals. Cytoplasmic determinants are molecules in the cytoplasm of a cell that help to determine cell fate. The polarized distribution of such molecules in a mother cell can result in their inheritance by only one of the daughters following cell division, thus promoting differentiation of the daughter cells. This strategy is used frequently in early development, when maternal gene products localized to particular regions of the egg, are asymmetrically distributed to different blastomeres during cleavage. Induction is a process whereby one cell or group of cells can influence the developmental fate of another, and is a common strategy to control differentiation and pattern formation in development. Induction involves cell-cell signaling and can occur over various ranges. Responding cells may show a single stereotyped response to the inductive signal, or a graded response dependent on its concentration, in which case it is called a morphogen. Induction relies both on the ability of the inducing cell to produce the signal and the ability of the responding cell to receive the signal and react in the appropriate manner. The ability of the responding cell to react to an inductive signal is termed competence. The loss of competence is one mechanism by which cells become irreversibly committed to a given developmental pathway. ‘Two types of induction can be distinguished on the basis of the choices available to the responding cell. Instructive induction occurs when the responding cell has a choice of fates, and the inductive signal instructs the cell to adopt one of those fates in preference to the other. Permissive induction occurs when the responding cell is already committed to a certain developmental pathway, but needs the inductive signal to continue towards differentiation. These are special types of induction in which single cells behave differently to cell populations. In lateral inhibition, differentiated cells arise in a regularly spaced pattern within a group of equivalent undifferentiated cells due to random fluctuations in levels of a ubiquitous14 Related topics Section A ~ rst prin signal that inhibits differentiation. In the community effect, populations of cells can change their collective fate by secreting enough of an inductive signal to reach a critical concentration threshold, whereas isolated cells follow a different developmental pathway because they cannot produce enough of the signal on their own. Cell fate and commitment (A2) Mosaic and regulative development (A4) Maintenance of differentiation (A5) How cells differentiate Cytoplasmic determinants The specialized properties of different cell types are conferred by the proteins they contain. The process of differentiation must therefore involve the synthesis of different sets of proteins in different cells. Exceptionally, this may be achieved by DNA rearrangement, as in the differentiation of antibody-producing blood cells (Topic C1). However, most cells contain the same DNA, and different sets of proteins are made by the selective expression or activation of particular gene products. The synthesis of a functional protein is dependent on a series of steps including transcription, RNA processing, protein synthesis and post- translational protein modification (Topic C1). Any or all of these stages can be regulated, so differentiation usually begins with the activation of a particular regulator molecule, such as a transcription factor. What is of interest to develop- mental biologists is how such regulator molecules are controlled and how this fits in with the concept of developmental commitment as discussed in Topic A2. ‘There appear to be two major strategies for establishing commitment and hence initiating the series of events that results in cell differentiation: the inheritance of cytoplasmic determinants and the perception of external inductive signals. These strategies are discussed in more detail below. A cell can divide to produce two daughters committed to different fates in the absence of any external influences. Stem cells provide an excellent example of this process. Each division of a stem cell produces one daughter cell committed. to differentiate, and a replacement stem cell. This stereotyped division program can occur in isolation, indicating that the mechanism of differentiation is entirely intrinsic. One way in which this could be achieved is through the asym- metric distribution of cytoplasmic determinants (molecules in the cytoplasm that can help to determine cell fate). If a mother cell contains a cytoplasmic determinant that is localized to one pole as the cell undergoes division, that determinant will be inherited by only one of the daughters. In the simplest scenario, the determinant could be a transcription factor that would activate certain genes in only one daughter cell (Fig. 1). Cytoplasmic determinants are encountered in many developmental systems. One example is the Drosophila nervous system, where stem cells divide to produce further stem cells and progenitor cells that are committed to differen- tiate into neurons. This reflects the asymmetric distribution of at least two proteins (a transcription factor called Prospero and a signaling protein called Numb) which are inherited by the neuronal progenitor. Further discussion of this system can be found in Topic J5. Many examples of segregating cytoplasmic determinants are found in early animal development, where maternal gene products are used as determinants toA3 ~ Mechanisms of developmental commitment 15 Induction O Inductive O Cytoplasmic | signal i cnet A “sO O @® oO Oo O Fig. 1. Cytoplasmic determinants and induction. polarize and pattern the early embryo. Many animal eggs are polarized with respect to the animal-vegetal axis, reflecting the distribution of the yolk and the consequent displacement of the nucleus towards the animal pole (Topic F1), but there is often additional hidden polarity at the molecular level. For example, the maternal mRNA for the transcription factor VegT is localized to the vegetal pole of the Xenopus egg. Active VegT is therefore synthesized only in the vegetal cells in the blastula-stage embryo, and this plays an important role in early patterning, specifically in mesoderm /endoderm specification and the formation of the embryonic organizer (Topic K1). The distribution of maternal mRNAs in the Drosophila egg is critical in the establishment of the anteroposterior axis. Maternal bicoid mRNA is located at the anterior pole and maternal navi0s mRNA located at the posterior pole. The proteins encoded by these messages control the establishment of gradients of transcription factors in the early syncytial embryo, which in turn control downstream genes that divide the embryo into segments and confer upon each segment its positional identity (Section H). The role of maternal gene products in early development is discussed in more detail in Topic Ad. Two identical cells can follow alternative fates if one is exposed to an external signal (often secreted by a different cell) while the other is not. The process where one cell or group of cells changes the developmental fate of another is termed induction and animal development is rich in examples of such induc tive interactions. The inductive signal may be a protein or another molecule secreted from the inducing cell. It usually interacts with a receptor on the surface of the responding cell (although some inductive signals diffuse through the membrane and interact with cytosolic receptors). The signal initiates a signal transduction cascade inside the responding cell that alters the activity of tran- scription factors and/or other proteins, and eventually alters the pattern of gene expression (Topic C3). Unlike the segregation of cytoplasmic determinants, induction is an extrinsic process that depends on the position of a cell in the embryo (Fig. 1). Induction can occur over different ranges (Fig. 2). In the most extreme case, endocrine signaling involves the release of inductive signals by one type of cell located a considerable distance from the responding cell population, and the molecule must travel to its target through the vascular system. This is the mech- anism utilized by many hormones. Most inductive interactions in early devel- ‘opment occur locally. This can involve the release of a diffusible substance (paracrine signaling) or a substance that is deposited in the extracellular matrix.16 Section A - First principles Signaling Responding cell cell Vascular system ain Single response QO. — Morphogen gradient Reciprocal Fig. 2. Ranges and types of induction. The range of such signals varies, but rarely exceeds a few cell diameters. The range of a signal can be extended by a relay mechanism, in which one signal causes the responding cell to release another signal, which similarly induces a third and so on. It is therefore possible for the responding tissue to take on the inductive properties of the inducer, a phenomenon termed homeogenetic induction (e.g. see Topic J1 for a discussion of homeogenetic induction in the vertebrate nervous system). In other cases, the signal may only reach the cell adjacent to the inducer, particularly in cases where direct contact between cells is required because the inductive signal is fixed to the cell membrane, or it travels through gap junctions (juxtactine signaling). Cells may also reciprocally induce each other, i. each cell acts as the inducer of the other, and responds to the other's inducing signal. Such reciprocal induction occurs in the kidney, where the ureteric bud induces the metanephric blastema to differentiate into the adult kidney structures, while the blastema induces the ureteric bud to proliferate and branch. This system is discussed in more detail in Topic K4. An important distinction can be drawn between inductive signals that elicit a single, stereotyped response in responding cells, and those that elicit different responses at different concentrations. The former type of induction is prominent in situations where two tissues come together for the interaction to take place, eg. in neural induction (Topic J1). The latter type of inductive signal is known asa morphogen'. Morphogens are of great significance in development because a morphogen gradient can be set up within a given tissue, and different responses can be induced depending on the distance from the source of the morphogen. This is one way in which cells can receive positional information, "Do not confuse a morphogen with morphogenesis. Morphogenesis is the process by which structures form during development, reflecting different types of cell behavior. A morphogen is an inducer that elicits different responses at different concentrations; the term literally means ‘form-generating substance’A3- Mechanisms of developmental commitment 7 Competence induction Lateral inhibition and the community effect and differentiate according to their position along an axis. The different, concen- tration-dependent responses in terms of alternative patterns of gene expression give cells positional values that enable them to form the correct structures in the body plan. This subject is considered in more detail in Topic A6 Competence is a property of the cell responding to induction. A cell is described as competent if it can respond to the inductive signal by undergoing all the appropriate molecular changes that allow it to follow the ‘induced’ develop- mental pathway. In the absence of induction, the cell eventually becomes deter- mined to an alternative pathway, and this coincides with its loss of competence to respond to induction (Topic A5). In the case of endocrine or paracrine signaling, competence depends on the synthesis of all the components of the signal transduction pathway that link the inductive signal to its target, such asa transcription factor, in the responding cell. If any of these components is lost, e.g. the cell surface receptor, the signal transduction apparatus, or the down- stream target transcription factor itself, the cell loses competence. In the case of juxtracrine signaling, a cell may also lose competence simply by breaking contact with the inducing cell. This may reflect the movement of cells away from each other, or the disassembly of gap junctions, These two categories of induction reflect the choices available to the responding cell. Instructive induction occurs where the responding cell has a choice of fates and will follow one pathway in response to induction but an alternative pathway in the absence of induction. For example, in the early Xenopus blastula, ectoderm will form epidermis in the absence of induction, but neural plate in the presence of inductive signals from the notochord. Permissive induction occurs where the responding, cell is already committed to a certain develop- mental fate, and simply requires the inducing signal to continue down that developmental pathway. An example is muscle development, where myoblasts continue to proliferate until growth factors are withdrawn, when they differen- tiate into myotubes and eventually muscle fibers. Growth factor withdrawal prevents the synthesis of an important class of inhibitor protein, and allows expression of the transcription factor MRF4, which drives muscle differentia- tion. The molecular basis of muscle differentiation is discussed in more detail in Topic K3. This example also demonstrates that induction may occur just as well through the withdrawal of an existing signal as it does through the secretion of a new signal, Instructive and permissive induction can be distinguished by grafting experi- ments. For example, in mammals, the cardiogenic mesenchyme (future heart) is required to induce hepatocyte development in the presumptive liver-forming, region of the foregut. The signals could be instructing the foregut to form hepa- tocytes instead of other cell types or could be permitting the differentiation of hepatocyte cells that have already been specified by other mechanisms. If the cardiogenic mesenchyme is grafted under the hindgut, no hepatocytes are induced. The inductive signals from the cardiogenic mesenchyme are therefore permissive rather than instructive (Topic K6).. Lateral inhibition is the inhibition of a particular developmental process in one cell by signals from an adjacent cell. Lateral inhibition can be used as a special form of induction, which involves an initially equivalent field of cells, yet results in the differentiation of individual cells ina regularly spaced pattern (Fig. 3).18 Section A - First principtes betel ger! = __ i Pe RERIEp aaa ee How spacing patterns are generated by lateral inhibition. Since all the cells are initially equivalent, they all have the potential to differen- tiate in the same way. However, in the undifferentiated state, they all signal to each other to repress differentiation. As individual cells begin to differentiate, their ability to repress the differentiation of neighboring cells increases while their tendency to be repressed diminishes. This could be achieved, for example, by increasing the production of the inhibitor signal and decreasing the synthesis of its receptor as part of the process of differentiation. The cells that happen to produce more of the inhibitor signal through random fluctuation would there- fore begin to differentiate and suppress the differentiation of the cells around them by producing more of the inhibitor signal. The spacing, of the differenti- ated cells would be governed by the range of the signal and the strength of its effect (Fig. 3). Lateral inhibition is thought to control the choice of neuronal progenitors in Drosophila and vertebrates (Topic J5), the choice between alterna- tive cell fates in the nematode vulva (Topic L1) and perhaps the distribution of trichomes on the leaves of flowering plants (Topic N5). The community effect is a mechanism whereby a population of cells must be present to change the collective cell fate (Fig. 4). This special form of induction community effect concentration of inducer is high — differentiation Oo o no differentiation Q © no community effect concentration of inducer is tow Fig. 4. The fate of inalvidual cells and tissues may differ due to the community effect,Ag - Mechanisms of developmental commitment 19 involves autocrine signaling, where the cell secretes a signal that acts on one of its own receptors. A single cell in isolation does not produce enough of the signal for an inductive effect to occur, because the signaling molecule does not reach sufficient concentration to trigger the effect. However, a population of cells produces enough of the signal, with the result that the fate of the entire population is changed en masse. The community effect occurs during both mesoderm induction (Topic K1) and neural induction (Topic J1) in Xenopus. For example, isolated animal cap (ectoderm) tissue from the Xenopus blastula-stage embryo will differentiate into epidermis, whereas individual cells can form neurons. This is because the ectoderm secretes certain bone morphogenetic proteins that inhibit neural differentiation, but isolated cells do not produce enough of the signal to prevent this outcome.Section A - First pi A4 MOSAIC AND REGULATIVE DEVELOPMENT Key Notes Related topics Cytoplasmic determinants and inductive signals can both be used to control cell fates during development. The exclusive use of either mechanism defines two extreme strategies, mosaic development and regulative development, Most organisms use a combination of these strategies. Mosaic development is controlled entirely by cytoplasmic determinants. Each cell undergoes autonomous specification, so that its fate is determined by its lineage irrespective of its position. Isolated blastomeres develop into the complete corresponding parts of the embryo as shown by the fate map, and embryos with missing blastomeres develop with the corresponding missing, parts. Regulative development is controlled entirely by inductive interactions. Each cell is specified conditionally, according to its interactions with other cells. Cell fate is therefore determined by position, irrespective of lineage. Isolated blastomeres do not develop into the corresponding parts of the embryo (as defined by the fate map) because they lack the appropriate interactions. However, embryos with missing blastomeres can regulate to replace their missing parts. Most species use a combination of mosaic and regulative mechanisms in their developmental program, Certain animals, such as ascidian, rely on cytoplasmic determinants for many aspects of early development and produce mosaic-like embryos. Conversely mammals appear to possess no determinants, and early development is entirely regulative. Other animals fall between these extremes, with both cytoplasmic determinants and inductive interactions playing important roles in early development. The development of plant embryos and meristems is also highly regulative. Matemal gene products are often used as cytoplasmic determinants, and these are placed in the egg during oogenesis. In such cases, early development is controlled by the maternal genome, and developmental mutations show a maternal effect (i. the phenotype of a mutant individual is seen in her offspring). Zygotic genes become active during, development, and this may be a more or less gradual process depending on the species. Mechanisms of developmental Molecular aspects of embryonic commitment (A3) pattern formation in Drosophila Early animal development by a) single cell specification (G1) Development of the plant embryo Barly patterning in vertebrates (2) ay Shoot and root meristems (N4)Ag ~ Mosaic and regulative development a Definitions of mosaic and regulative development Mosaic development Egg contains cytoplasmic. determinants Cytoplasmic determinants and inductive signals can both be used to control cell fates (Topic A3). If development was controlled entirely by cytoplasmic deter- minants, the fate of every cell would depend on its lineage, while its position in the embryo would be irrelevant. This is the definition of mosaic development. Conversely, if development was controlled entirely by inductive interactions, the fate of every cell would depend on its position in the embryo and its lineage would be irrelevant. This is the definition of regulative development. The development of most organisms involves a combination of these mechanisms. The two mechanisms are discussed in more detail below, and their key features are compared in Fig. 1. Properties of mosaic embryos In mosaic development, the fate of every cell is governed entirely by its intrinsic characteristics, i.e. the cytoplasmic determinants it inherits at cell division, and fate is therefore equivalent to potency. Cell fate depends on lineage (line of descent) rather than position in relation to other cells. During development, each cell is said to undergo autonomous specification, ie. if removed from the embryo each cell should, in principle, develop according to its intrinsic instrac- tions and differentiate into the appropriate part of the embryo whether or not the rest of the embryo is there. Furthermore, the remainder of the embryo should also develop normally, but would lack the parts specified by the missing cell (Fig. 1). The fate map of an embryo undergoing mosaic development can therefore be constructed by looking at the fate of individual cells developing in isolation. Formally, the fate map is equivalent toa specification map (Topic A2). Removal of region B from fate map ‘Segregation of determinants by cell division Regulative Egg contains no determinants Goneraiion o Progressive inductive initia asyrmety Inleractons e.g. by sperm entry & +o SF removal of roglon 8 from fate map Fig. 1. Behavior of embryos and explants during mosaic and regulative development.Section A ~ First principies Regulative development Prevalence of mosaic and regulative development Problems with mosaic development as a sole strategy Many animals use mosaic mechanisms in early development but it is not practical to base the entire developmental program on this strategy unless the organism is very simple. For even moderately complex organisms, the egg would need to be divided into thousands of zones that would eventually segre- gate into different cells. Furthermore, continuation between generations would require the gamete forming region of the embryo to be predetermined in the egg, and prepatterned with the cytoplasmic determinants of the subsequent generation. Each generation would have to be packaged and patterned in the gamete-forming region of the last, like a series of Russian dolls, in a manner iscent of preformation (Topic A1). remii Properties of regulative embryos In regulative development, the fate of every cell is governed entirely by its interactions with other cells. Cell fate depends on position in the embryo and is independent of lineage. The potency of each cell is therefore much greater than its fate. During regulative development, each cell is said to undergo conditional specification, i.e. conditional on the presence of other cells. Therefore, removed from the embryo, a given cell will not fulfil its normal fate because it lacks the necessary interactions. Furthermore, the remainder of the embryo can regulate to replace missing parts, because the appropriate inductive interactions have yet to take place, and other cells can be respecified to fill in the missing pattern (Fig. 1). The fate map of a regulative embryo is not the same as a specifi- cation map, since cells in isolation will not develop in the same way as those in the embryo. Problems with regulative development as a sole strategy Regulative development is suited to a developmental program based on epi- genesis, because sequential inductive interactions could progressively increase the complexity of an embryo by increasing the number of cell types. One problem with regulative development, however, is starting the process off, since development begins with a single cell. Regulative embryos need an alternative process to introduce asymmetry into the system and set up the initial source juctive signals that sets off the cascade of conditional specification. This asymmetry could be generated by cytoplasmic determinants, or it could come from an external source. Vertebrate embryos provide examples of both. In Xenopus, for example, there are asymmetrically-distributed maternal gene products in the egg, which act as cytoplasmic determinants, while the site of sperm entry provides an external symmetry-breaking cue. These signals help to establish the initial source of inductive signals, which is called the organizer. The establishment of the organizer is discussed in detail in Section I, and in the context of mesoderm induction, in Topic K1 All organisms use both cytoplasmic determinants and inductive interactions during development, with some cells specified autonomously and some condi tionally. The predominance of each strategy varies in different species. In ascid- ians, the epidermis, endoderm and most muscle cells are specified by the distribution of cytoplasmic determinants in the egg, so that embryos show striking mosaic behavior. This is most obvious in species such as Halocynthia roretzi, where the determinants and the tissues they give rise to are different colors (Topic G3). However, the specification of other tissues, such as theAg - Mosaic and regulative development 23 Maternal and zygotic genes notochord and central nervous system, is conditional upon cell-cell interactions. In contrast to ascidians, the early development of mammals is purely regulative. There appear to be no determinants in the egg, so each of the blastomeres in the morula is equivalent and totipotent. The first differentiation separates the inner cell mass from the outer trophectoderm, and this appears to be dependent only on position within the morula, with those outer cells exposed to the environ- ment differentiating into trophectoderm (Topic F1). Cytoplasmic determinants appear much later in mammalian development, e.g. during neuronal differenti- ation (Topic J5). The development of plant meristems is also highly regulative (Topic N4). Insects employ a specialized strategy, syncytial specification, where the egg nucleus divides a number of times to generate a syncytium containing thousands of nuclei. Cell fates are determined prior to cellularization by interac- tions between diffusible regulators and individual nuclei. Drosophila develop- ment is illustrative of this strategy and is described in Section H. Cytoplasmic determinants often feature in early animal development, where maternal gene products are localized in the egg to help pattern the early embryo. in such cases, early development becomes dependent on the maternal genome, while the paternal genome plays no part. This leads to an interesting, class of mutants called maternal effect mutants, whose phenotypes are manifest, in the embryos of mutant females, but not in the mutant females themselves. Furthermore, if the mutation is recessive and the female is homozygous for the mutation, embryonic development cannot be rescued by mating to a wild type male. Maternal gene products may be critical in the earliest stages of development, but they may be active for only a short time, or they may become depleted. Development then becomes dependent on zygotic gene products, and zygotic mutations that affect development become apparent. The beginning of zygotic gene expression can be a landmark in development, accompanied by changes in the cell cycle (Topic C4) and in cell behavior. In Xenopus, this is called the midblastula transition and occurs just prior to gastrulation (Topic Fl), A similar landmark occurs in Drosophila development, coinciding with the cellularization of the blastoderm (see Topic C4 for a discussion of cell cycle regulation in this system). However, unlike the Xenopus midblastula transition, the sudden change in Drosophila development does not mark the onset of zygotic gene expression, but rather the depletion of one or more key maternal gene products that regulate the cell cycle. In fact, zygotic gene expression in Drosophila commences gradually, as it does in most species. In mammals, zygotic gene expression begins during early cleavage, as early as the two-cell stage in some speciesSection A - First principles A5 MAINTENANCE OF DIFFERENTIATION Key Notes Genomic sauivatence| [Potency of differentiated cells. Maintenance of differentiation With few exceptions, all differentiated cells in a given organism contain, the same DNA as the fertilized egg. This means that most differentiated cells possess the information required to produce an entire organism. Although they carry all the necessary DNA, differentiated animal cells cannot recapitulate the developmenial program and produce a new animal. Conversely, differentiated plant cells can regenerate whole plants, either by the direct production of shoots and roots, or by somatic ‘embryogenesis. This shows that the differentiation of animal cells is generally irreversible, although there are some examples of limited plasticity, such as the regeneration of limbs in urodele amphibians. “Totipotency of nuclei] Nuclei from differentiated animal cells can support development when injected into an enucleated egg, although the process is more efficient using embryonic nuclei than nuclei from adult cells. This confirms that nuclei retain all the genetic information required for development, but suggests the DNA is modified in some way as cells differentiate. Cell fate in animal development is progressively restricted and usually irreversible. This means that animal cells retain a memory of previous developmental decisions, which is perpetuated through successive cell divisions. Both cytoplasmic and nuclear mechanisms are involved in developmental memory and the maintenance of differentiation, Transcription factors can regulate their own production to maintain the expression of a particular set of genes. Furthermore, by controlling, chromatin structure and the extent of DNA methylation, active and repressed chromatin domains can be maintained through successive cell divisions. Plants may use similar mechanisms to regulate gene expression, but their developmental memory can be erased by placing cells ina new environment. Related topics Cell fate and commitment (A2).__Somitogenesis and patterning (K2)_| Mechanisms of developmental Plant 7s animal development (NI) | commitment (A3) | Chromatin and DNA methylation (C2) Genomic With very few exceptions, all differentiated cells in a given organism contain the equivalence same DNA, which is the same as the DNA in the fertilized egg, This means that almost all differentiated cells in principle contain all the information required to generate a complete new individual. Evidence for genomic equivalence comesAS - Maintenance of differentiation 25 Potency of ifferentiated cells from molecular analysis including Southern blot hybridization and PCR amplification of genomic DNA isolated from embryos and various adult tissues. When probed for different developmental marker genes and cell type- specific genes, all banding patterns and PCR products are the same. There are a few notable exceptions to this rule, which are listed in Table 1 Table 1. Examples of genomic non-equivalence during development Mechanism Examples DNA amplification Selective amplification of rRNA genes in Xenopus oocytes. Amplification of the chorion genes during oogenesis in Drosophila (Topic E3). DNA loss In mammals, loss of nuclei in differentiated erythrocytes and keratinocytes. DNA rearrangement —_In vertebrates, VDJ recombination and class switching during B- and T-cell development (Topic C1) If all differentiated cells carry the same genetic information, it should be possible in principle to use differentiated cells to generate new individuals. In plants this is generally true. Differentiated plant cells can be isolated and will, under the appropriate culture conditions, form undifferentiated tissue called callus. This can be used to regenerate whole plants, either by organogenesis (direct formation of shoots and roots) or somatic embryogenesis (full recapitu- lation of development, including the embryonic stage). This makes it possible to produce whole plants from cuttings, callus or even single cells in culture, and has greatly facilitated the production and exploitation of transgenic plants (Topic B3). Conversely, differentiated animal cells are almost always irreversibly committed to a particular fate, and as development proceeds, this fate becomes more and more restricted until the cell is terminally differentiated (Topic A2) There are certain cases in which cell fate in animals shows a limited plasticity. Drosophila imaginal discs are undifferentiated larval structures that produce specific appendages in the adult fly (Topic L2). Their fates are usually deter- mined during embryogenesis, but if larval discs are serially cultured in the abdomen of adult flies, they can undergo transdetermination and later differen- tiate to form alternative structures. As the imaginal discs represent an early stage of commitment, it is not really surprising that their fates can be altered when the cells are continuously exposed to such an artificial environment. A more striking example of developmental plasticity is the ability of urodele amphibians to regenerate limbs and tails (Topic M3). In this case, fully differen- tiated cells at the wound site can dedifferentiate to give pluripotent progenitor cells, which then redifferentiate to form the various different types of replace- ment cells. This process can be termed transdifferentiation. Although the transdetermination of insect imaginal discs and the transdiffer- entiation of cells in the urodele limb represent examples of how differentiated cells can reverse their fate and recapitulate at least part of the earlier develop- mental program, these cells are still unable to give rise to an entire new animal. This indicates that animal cells remember developmental decisions taken by their ancestors and, except in the limited cases discussed above, their fates are fixed by mechanisms that maintain differentiation.Section A - First principles Totipotency of nuclei Maintenance of differentiation Although differentiated animal cells cannot recapitulate the entire develop- mental program, it is possible for the nuclei from those differentiated cells to do so. This type of experiment, where the nucleus of a differentiated cell is used to replace the nucleus of a fertilized egg, shows that all the information required to generate the animal is retained in the nuclei of differentiated cells. The earliest experiments of this kind were performed on Xenopus. Nuclei taken from tadpole cells or from different kinds of adult cell were injected into UV-irradiated eggs (UV treatment destroys the endogenous DNA). In many cases, the eggs containing tadpole nuclei developed into normal swimming tadpoles, and in a few cases these produced viable adults. Adult cell nuclei from many different cell types were also able to support full development but at a much lower efficiency. However, nuclei from some adult cell types consistently failed to allow development. The results from such experiments firstly confirm the totipotency of adult cell nuclei, showing there is no irreversible change to genetic information in the nucleus during development. However, it becomes more difficult to recapitulate the entire developmental program as development proceeds, and indeed becomes impossible in certain cell types, such as neurons. Thus, although there is no change to the genetic information in most differenti- ated nuclei, the DNA does undergo some change that reduces the ability of the nucleus to be reprogrammed by the intracellular environment of the egg. These results have been confirmed in other species, including insects and mammals. It has been possible for a long time to produce mammals by nuclear transfer from other embryos, but only recently has it been possible to produce e.g. sheep and cows by nuclear transfer from adult cells. There appear to be certain poorly- understood species-specific properties that affect the success of nuclear transfer procedures. For example, the technique as currently used is very inefficient in mice and pigs. The results of experiments using differentiated cells and their nuclei suggest that the differentiated state is maintained by a combination of cytoplasmic and nuclear factors. The role of the cytoplasm in differentiation is demonstrated by nuclear transfer experiments, where the egg cytoplasm is sometimes able to reprogram the nucleus of a differentiated cell to behave in the manner of an egg nucleus. Similarly, cell fusion experiments demonstrate that the cytoplasm from one cell can influence the nucleus of the other. For example, fusion between virtually any cell type and a muscle cell will induce the expression of muscle- specific genes in the non-muscle cell nucleus; this is a limited type of transdiffer- entiation. The cytoplasm is the site of protein synthesis, so it is likely to be full of newly synthesized transcription factors awaiting import into the nucleus. The arrival of a new nucleus therefore attracts these transcription factors to the sites they would occupy in their own nucleus, inducing similar patterns of gene expression. This would be particularly true in the egg, which has a larger volume of cytoplasm than any other cell, and a stockpile of proteins. The maintenance of differentiation through successive rounds of cell division could therefore be achieved by transcription factors. In one mechanism, the production of large amounts of a given transcription factor would provide enough of the regulator to persist in the cytoplasm through numerous cell divi- sions. A more refined mechanism would be for the transcription factor to exer- cise positive feedback regulation on its own gene. This appears to be the case in muscle differentiation, where the transcription factor MyoD activates not only a range of muscle-specific genes encoding structural proteins, but also its ownAS ~ Maintenance of differentiation ar gene and the genes for other muscle-specific transcription factors (Topic K3). In principle, however, this type of cytoplasmic loop could be broken by transfer ring the nucleus toa different cytoplasm. The role of the nucleus in differentiation is demonstrated by the inability of nuclei from certain cell types to recapitulate the developmental program in the egg, and the decrease in the efficiency of recapitulation as cells become progres- sively more differentiated. As discussed in Topic C2, both DNA methylation and chromatin structure have profound effects on gene expression, and there are mechanisms for perpetuating both types of modification through successive rounds of DNA replication. Hence, in female mammals, the X-chromosome inactivated in one cell remains inactive in all descendants in part because the DNA methylation pattern is perpetuated by DNA methyltransferases that pref- erentially recognize hemimethylated DNA (DNA methylated on one strand only). In Drosophila it has been shown that homeotic selector gene expression patterns are initiated by transcription factors encoded by the upstream gap genes, but the patterns are maintained through successive rounds of cell divi- sion by reorganization of chromatin structure, mediated by the Polycomb and trithorax gene products (Topic H5). Related genes have been identified in vert brates, and their products maintain Hox gene expression Differentiated plant cells use transcription factors to regulate gene expression, and in the case of Arabidopsis, it has been shown that homeotic selector gene expression is also maintained by chromatin reorganization. As plants and animals use similar mechanisms of gene regulation, how do differentiated plant cells remain totipotent? Developmental responses in plants, such as the growth of shoots and roots and the production of callus tissue, can all be elicited by treatment with different hormones. This suggests that the differentiated state of the plant cell is maintained predominantly by hormonal signaling, and that despite their similarities, maintenance mechanisms involving. transcription factors or DNA/chromatin modification in plants are inherently reversibleSection A - First principles A6 PATTERN FORMATION AND COMPARTMENTS Key Notes Regional specification] ‘Compartments and” "segmentation | Homeotic genes and ‘mutations Regional specification describes any mechanism that tells a cell where it is in relation to other cells in the embryo. Regional specification is necessary for pattern formation, where cells become organized correctly and generate appropriate structures in different parts of the embryo. Pattern formation begins with a basic body plan. The organization of cells to form a body plan requires a polarized framework of coordinates, represented by the principal body axes. In animals these are the anteroposterior, dorsoventral and left-right axes. The axes are specified and polarized either by the localization of cytoplasmic determinants, or the perception of external physical cues. The location of any cell in the embryo can be defined by its position along each of the principal axes. A key question in developmental biology is how cells become aware of their position, ie. what is the nature of the positional information they receive? Several model systems suggest that cells may be assigned positional values on the basis of their distance from the source of a morphogen. A morphogen is an inductive signal that elicits different responses at different concentration thresholds. A morphogen synthesized at one end of an axis would establish a concentration gradient along the axis, and cells could be assigned positional values according to the concentration of morphogen. The gradient of morphogen would be translated into a gradient of intracellular signal transduction and ultimately differential gene expression. Morphogens can establish gradients by free diffusion but alternative mechanisms are required for extracellular secreted proteins such as Hedgehog, Wingless /Wnt and Decapentaplegic/BMP. Once an axis has been established, it is often divided into repetitive units that allow the same basic developmental structure to be used in different ways. These units may exist only in terms of boundaries of gene expression, or they may become manifest as physical structures such as segments. They are often developmental compartments, i.e. structures within which the clonal expansion of a particular cell line is constrained Homeotic genes give cells their positional identity. Different combinations of homeotic genes are expressed in response to different morphogen concentrations, and in this way cells can be assigned positional values along an axis. The homeotic genes encode transcription factors that regulate downstream effector genes controlling, differentiation and morphogenesis. Homeotic mutations cause cells to beAG ~ Pattern fermation and compartments 29 Related topics assigned incorrect positional identities, resulting in the development of one body part with the likeness of another. There is evidence for spontaneous pattern formation in systems such as the developing limb and plant meristems, since these can form normal organs when disaggregated, and extra organs when oversized, whereas in both cases they would be expected to produce disorganized tissues. Patterns in developing organs are likely to be built up in layers, starting with a weak pre-pattern that is refined and strengthened by later acting genes. Axis specification and patterning Mesoderm induction and patterning, in Drosophila (Section H) (KI) Axis specification in vertebrates Somitogenesis and patterning (K2) Gection 1) Patterning and morphogenesis in Patterning the anteroposterior limb development (M2) neuraxis (2) Flower development (N6) Patterning the dorsoventral neuraxis (]3) First coordinates Regional specification is any mechanism for telling a cell where it is in relation to the rest of the embryo, so that it can behave in a manner appropriate for its position. Regional specification allows cells to adopt the correct spatial organi- zation, and is therefore an essential requirement for pattern formation, the process by which cells act on their instructions and become organized into appropriate structures. In some cases, pattern formation involves telling a cell to undergo the correct form of differentiation. For example, hepatocytes should differentiate only in the liver-forming region of the foregut and nowhere else. In other cases, the same cell type is found throughout the body, but the cells must behave differently at different positions in order to generate regionally appro- priate structures. For example, bone tissue is found throughout the vertebrate spinal column, but the architecture differs according, to position so that ribs form only in the thorax and the atlas and axis form only in the neck. Development is progressive (Topic A1), so pattern formation occurs step by step, starting with an initially simple body plan that is gradually filled with detail. The establishment of a body plan requires each cell in the embryo to be aware of its position in relation to other cells, and this in turn requires the defin- ition of a basic framework for positional coordinates. The framework comprises the principal axes of the embryo, All vertebrates and many other animals share a similar basic body plan that is, at least superficially, bilaterally symmetrical The central nervous system runs along this plane of symmetry and defines the anteroposterior axis. In vertebrates, the same axis may be termed the cranio- caudal or rostrocaudal axis, literally meaning ‘head to tail’, so that it can be applied equally to fish, birds and terrestrial vertebrates whether they walk on. four legs or upright. The other principal axis, the dorsoventral axis, runs from back to belly; the dorsal side faces upwards and the ventral side downwards, ‘Once the anteroposterior and dorsoventral axes are in place, the remaining axis, the left-right axis, is specified by default The names of all three principal axes infer polarity, i.e. the anteroposterior axis rans from the anterior end of the embryo to the posterior end. The brain isSection A - First principles Positional information Morphogen gradients found at the anterior end, and this is usually the end of the animal that moves forwards. Polarization is an important part of axis specification and requires a symmetry-breaking process, such as the asymmetric distribution of cytoplasmic determinants in the egg, or an external physical cue. For example, the antero- posterior axis of the Drosophila embryo is polarized by the distribution of maternal gene products (Section H). The anteroposterior axis of the vertebrate embryo, however, is polarized by a physical cue, such as gravity in the case of chickens (Topic I1). The left-right axis is established by default, but must still be polarized. This appears to involve an intrinsic mechanism based on molecular asymmetry (Topic I3). In particular developmental systems, the principal axes may have different names. When considering the development of individual structures such as limbs or eyes, the proximodistal axis refers to the axis running from the body towards the extremity of the structure (see Section M). Animal eggs have an animal-vegetal axis if the nucleus is displaced to one end; this often occurs if the egg is yolky, as in amphibians, reptiles and birds (Topic I1). Plant embryos have an apical-basal axis, which corresponds to the shoot to root axis of the seedling (Topic N2). Leaves have both apical-basal and dorsoventral axes, the dorsal side facing upwards towards the sun (Topic N5), ‘Once the principal axes of the embryo have been established, the location of any cell in the embryo can be absolutely defined according to its position along each of the axes, rather in the same way that a town can be found on a map by finding its coordinates. In this manner, each cell has an ‘address’ telling it where it is, and thus how to behave to generate regionally appropriate structures. This address is known as a positional value or a positional identity. An important question addressed by developmental biologists is how cells become aware of their positional identities. What is the nature of the positional information that tells a cell where it is in the embryo? Several experimental systems, including early Drosophila development and limb development, suggest that positional information may be generated by a morphogen gradient. A morphogen is a substance that can influence cell fate, but has different effects at different concentrations. In its simplest form, the morphogen model suggests that positional information along an axis can be generated by the synthesis of a morphogen at a source at one end of the axis, and diffusion away from the source would set up a morphogen gradient. Cells along the axis would receive different concentrations of the morphogen and this would induce different patterns of gene expression at different concentration thresholds (Fig. 1). Such % 12. . - — OOOOO0CCO0O, @OOOOCCOOO §=—@OOO|OOC|OOO ®t 2 3 @ Lines of cells, non polarized External signal specifies Cells respond to the concentration one cell as ‘posterior of the morphogen (T1, T2) and become by inducing the synthesis patterned along the anteroposterior of a morphogen axis (regions 1, 2 and 3) Fig. 1. Principle of a morohogen gradient.6 - Pattern formation and compartments 31 concentration-dependent patterns of gene expression would represent the ‘address’ or positional identity of the cell. ‘A number of systems have been studied that appear to involve morphogen gradients. In early Drosophila development, the Hunchback protein is distrib- uted in a gradient along the anteroposterior axis of the syncytial embryo. Hunchback is a transcription factor and activates downstream genes such as Kriippel, knirps and giant in a concentration dependent manner. The latter genes are expressed in stripes defining broad sectors of the anteroposterior axis (Topic H2). Similarly in vertebrate limb development, the signaling protein Sonic hedgehog is expressed in the posterior zone of polarizing activity (ZPA) of the limb bud, and establishes a concentration gradient running from posterior to anterior. Although the mechanism is complex (Topic M2) the result is that different HoxD genes are activated in concentric rings surrounding the ZPA, resulting in the formation of different digits from essentially the same cell types. In both these systems, interfering with the level and/or position of the morphogen has profound effects on development. The data suggest that such experimental manipulations cause cells to receive the wrong positional informa. tion, consequently they adopt the zerong positional identities, resulting in the appearance of regionally inappropriate structures. In the early Drosophila embryo, transcription factors can act as morphogens by diffusing freely through the cytoplasm to establish concentration gradients. Similarly, the lipid signaling molecule retinoic acid may be able to establish a concentration gradient across a field of cells by free diffusion. It is thought unlikely, however, that secreted proteins of the Hedgehog, Wingless/Wot and Decapentaplegic/BMP families, all of which function as morphogens in a variety of developmental systems, can establish concentration gradients in this manner. Recent evidence has emerged to suggest that such proteins could act as morphogens in a variety of ways (Fig. 2): © Initiating a signal relay, where one signaling protein diffuses over a short range, but induces adjacent cells to produce another signaling protein, which acts in the same way, with diminishing effect across a field of cells. For example, Sonic hedgehog has been shown to induce BMP2 expression in adjacent cells in the vertebrate limb. © Responding cells could extend cytoplasmic processes (cytonemes) to contact the inducing cell, and the longest cytonemes would be extended by more distant cells. Ligands binding to receptors on the end of the cytonemes would induce the same signal transduction pathway in all cytonemes, but the response to signaling might be diminished as a function of increased distance from the cell nucleus. Such cytoplasmic processes have been observed in Drosophila. © The signaling protein could be propagated by a carrier. Proteoglycans in the extracellular matrix have been implicated in this role, and mutations affecting proteoglycan synthesis such as sugarless and fout velw have been shown to inhibit Hedgehog and Wingless signaling in Drosophila. © The signaling protein could be transported from cell to cell by cyclical endo- cytosis and exocytosis. Evidence for the intracellular transport of Wingless has been reported in Drosophila. Finally, it should be noted that concentration-dependent induction can also be established by an ‘anti-morphogen’ gradient. For example, a morphogen can be secreted uniformly, but the level of its activity can be graded by the diffusion of32 Section A - First principles Compartments and segmentation Simple diffusion Signal relay Cytonemes Surface transport by proteoglycans ds A\r er Intracellulal transport by e\;e 8 cyclical endolexocytosis Fig. 2. Mechanisms by which secreted proteins could generate marphogen gradients, an inhibitor. This is apparent in systems such as the vertebrate organizer, which secretes molecules that inhibit BMPs and Wnts (Topic 12). Similarly, differential responses to a uniform concentration of inducer can be achieved by varying the levels of the receptor in the responding cells, However, this requires pre- existing polarity in the system. ‘The establishment of an axis is often followed by segmentation, so that the axis is divided into a repetitive series of similar but independent developmental units. This is known as metamerism. It allows the same basic structure, gener- ated by the same set of genes, to be used over and over again for different purposes, and allows evolution of new functions by adding or deleting segments, Segments are exploited by many species, from the obvious segmenta- tion of insect and annelid bodies, to the rhombomeres and somites of vertebrate embryos, and the repetitive organ primordia of flowers. However, the overt segments seen in the fully developed embryo or adult may not correspond toA6 - Pattern formation and compartments 33 Homeotic genes and mutations the underlying metameric units defined by the developmental program. For example, the Drosophila anteroposterior axis is patterned on the basis of paraseg- ments that correspond to the anterior two thirds of one segment and the poste- rior third of the next. Similarly, the vertebrae of the eponymous vertebrates are formed from the anterior compartment of one somite and the posterior compart ment of the next. A Drosophila parasegment or a vertebrate rhombomere can be viewed as a developmental compartment, i.e. a structure within which a clone of cells is constrained (this is termed lineage restriction’). Clonal analysis (Topic B4) can be used to determine when compartments form (Fig. 3). For example, a cell labeled in the early chicken hindbrain, before the rhombomeres become apparent, may give rise to a clone that spans two rhombomeres. Conversely, a cell labeled after the boundaries have formed gives rise to a clone that is restricted to a single rhombomere. In some cases this may occur simply because the labeled cell was centrally placed and the progeny did not spread far enough to reach the putative boundary. However, if the labeled cell was near the edge of the compartment, a sharp boundary forms between labeled and unlabeled cells. The term allocation means that a cell and its clonal descendants are confined to a compartment. Allocation may occur simply because there is a physical barrier to cell mixing. Alternatively, compartments may be defined by patterns of gene expression in the absence of any obvious boundary. A homeotic transformation occurs when one body part develops with the like- ness of another, indicating that the cells involved have the wrong positional identities and have therefore developed into a regionally inappropriate struc- ture. In Drosophila, large scale screens of mutagenized flies revealed occasional homeotic mutations, i.e. individuals carrying mutations that caused a homeotic transformation between segments. The existence of such mutant flies indicated a genetic basis for positional identity, and allowed the mapping and cloning of the homeotic selector genes involved. The Drosophila homeotic selector genes map to two clusters, and encode transcription factors with a. helix-turn-helix DNA-binding motif termed a homeodomain (the part of the gene encoding the homeodomain is called a ynpartmentalization Clones may span boundaries ~. i . FL) a) i tT 7 After compartmentalization Some clones form sharp boundaries a oe qi q . Y Fig. 3. Clonal analysis of developmental compartments. ‘Lineage restriction means the (physical) restriction of a cell lineage to a developmental compartment. It does not necessarily mean that the jie of the cell is restricted in any way.Section A - First principles homeobox). Similar clusters, known as Hox gene clusters, are found in all animals, suggesting a common genetic basis for pattern formation in the animal kingdom. Although homeobox genes are also found in plants, there are no clus- ters involved specifically with pattern formation. Rather, they appear to encode transcription factors involved in other functions, just as homeobox genes unre- lated to patterning are also found in animals. Homeotic genes involved in flower patterning encode another family of transcription factors characterized by a motif termed a MADS box. In animals and plants, non-expression or misexpression of homeotic genes can lead to homeotic transformations ‘The Hox genes and MADS-box genes are discussed in more detail later (see Topics H5, K2 and N6). The purpose of this introductory topic is to illustrate the principle of homeotic gene function using a simple model. This model is some- times termed the epigenetic address model, and shows how positional values could be established by a morphogen gradient and how those positional values could be altered by homeotic mutations. The model is shown in Fig. 4. It assumes that a morphogen is synthesized at the source, S, and establishes a gradient across a field of four equivalent cells (C1, C2, C3, C4) that eventually form segments with four different structures. The cells can respond to the morphogen gradient at four concentration thresholds (Tl, T2, T3, T4) by expressing one or more homeotic genes (H1, H2, H3, H4). Ata low concentra- tion threshold (T1) only H1 is expressed. At a higher concentration threshold, H2 is expressed. Note however, that H1 is also expressed because its concentra- tion threshold has been exceeded. Similarly, H3 and Ha are expressed at the higher thresholds T3 and TA. This establishes a pattern of gene expression that is nested at the source of the morphogen, ie. nearest the morphogen all four homeotic genes are expressed while furthest away, only one is expressed. Such nested patterns are characteristic of anteroposterior axis patterning in animal embryos (see Topics H5 and K2, and for a similar pattern in limb development, see Topic M2). In this way, each cell can be given an ‘address’ or positional iden- tity in the manner of a simple binary code, depending on whether each gene is on or off. Cell C4 has the code 1000, while cell C1 has the code 1111. Now consider what happens in a loss-of-function homeotic mutant, when Hé is not expressed. In this case, the code for cell C1 becomes 1110, the same as C2. The result is that both CI and C2 would have the same positional identity and would develop into the same structure, ie. there would be a transformation of segment C1 into C2. Such homeotic transformations have been identified in vertebrates, Drosophila and Arabidopsis. Also consider what happens when one of the genes is overexpressed. If H2 is expressed in all four cells, C3 and C4 become equivalent in terms of their positional identities, and segment C4 transformed into C3. Again, such gain of function homeotic mutations have been generated in Drosophila and other systems. Finally, consider the effect of overexpressing H4 in alll cells. In this case, some entirely novel codes are produced, such as 1001. The effects of this type of mutation are unpredictable, and can lead to lethality, disorganization or the development of novel struc- tures. The epigenetic address model is simple and it fits data provided by gene expression patterns and mutant phenotypes in some developmental systems. However, in nature, the situation is much more complicated. For example, homeotic selector genes also regulate each other so that the misexpression of one gene also alters the expression patterns of several others. In vertebrates, it appears that the levels of Hox gene expression as well as the patterns are impor-14 B Pp tt 4 mutant v 13 1p n H2 overexpression 14 13 mR n H4 overexpression 1% 13 12 af] QOCOG OOCO©H OOOOH OOOO He HS He Codes 1111 1110 1100 1000 iy 1110 1110 1190 1000 sn aio 110 1100 syst 11011001 iy Novel ? Loss of aberrant segments ? Lethal? Fig. 4. Mode! for the generation of positional values using homeotic genes, and the basis of homeotic transformations. Syiounsedwoa puke ueneuHO) Weped - OV36 Section & - First principles Pattern, pre- pattern and spontaneous pattern formation tant in conferring positional values. Such complex interactions are beginning to be unraveled and are discussed in more detail in Topics H5, K2 and M2. What does a cell do with its positional information? The fact that the homeotic genes all appear to encode transcription factors suggests that the effect of positional information is to regulate the expression of downstream genes, probably those whose function is to control cell differentiation and morpho- genesis to ensure that the correct structures are formed in the appropriate posi- tions. This does in fact appear to be the case where downstream effector genes have been found, but surprisingly few downstream targets of homeodomain transcription factors have been identified (Topic H5). From the discussion above it is obvious that genes play an important role in pattern formation, and in many of the developmental systems used as models of pattern formation (eg. vertebrate limb development and neurogenesis), genes with patterning roles have been identified. However, in cases where such genes are mutated, it is often found that certain elements of the pattern remain. This indicates that pattern formation occurs in stages, starting with a pre-pattern that is refined and strengthened by the major patterning genes. In the developing vertebrate limb, for example, there are three signaling centers that polarize the three limb axes (Topic M2). In mutants where two of these signaling centers are abolished and the third is no longer polarized, a certain degree of normal patterning still occurs, suggesting that an underlying pre-patterning mechanism exists to establish the organizing centers in the first place. There is also evidence that certain developmental systems, such as the verte- brate limb bud and the meristematic tissues of plants, have an intrinsic pattern- forming capacity that is independent of gene expression. This is revealed by the unexpected formation of normal structures when these tissues are disrupted and reorganized. For example, the mesenchyme cells of the limb bud can be disaggregated and randomly mixed, and then replaced within the limb ecto- derm. After a period of disorganized growth, these buds will give rise to normal looking digits in the absence of the gene expression patterns that accompany normal limb patterning. This may reflect an underlying reaction-diffusion ES bo = LA_Al (c) Activator IIIwy Fig. 8. (a) A reaction difusion model in which the activator (white) and inhibitor (gray) are initaly uniformly distributed across a field of cells, but spontaneously generate regular peaks of the activator. &) Ths is achieved if the activator stin- lates its own synthesis and that of the inhibitor, and the inhibitor diffuses away faster than the activator. (| ifthe size of the field increases, so does the number of activator peaks.AG - Pattern formation and compartments 37 mechanism, a chemical system comprising two or more interacting diffusible components that can generate spontaneous patterns. Given that many patterning systems depend on diffusion (e.g. morphogen gradients, lateral inhi- dition), it would not be surprising if other developmental mechanisms were also based on diffusion. A number of models based on this type of mechanism have been proposed. In one model, there is an activator and an inhibitor. The acti- vator stimulates its own synthesis (ie. it is autocatalytic) as well as that of the inhibitor, and the inhibitor suppresses the activator. If the inhibitor diffuses at a greater rate than the activator, a repetitive wave-like pattern forms sponta- neously with peaks of activator activity (Fig. 5). The system is initially unstable, with some peaks disappearing, but it eventually stabilizes into a regular pattern. Importantly, if the size of the field is increased, the pattern adjusts accordingly and extra peaks are generated. This may explain the formation of extra digits in oversized limb buds, and extra floral organs in oversized floral meristems, rather than the disorganized tissue that would be expected if each organ was established independently.Section A - First principles A7 MORPHOGENESIS Cell division j [ Geil size sind shape | ‘Cell fusion Celt death ] Cell adhesion | Related topics In the dynamic embryo, cells assemble into tissues and organs with many different shapes and structures, and this process is termed morphogenesis. Morphogenesis reflects different aspects of cell structure and behavior, including cell division, cell shape and size, cell-cell adhesion, interaction between cells and the extracellular matrix, cell fusion, and cell death. Cell division influences morphogenesis in two ways. The rate of cell division contributes to differential growth in different parts of the embryo, while the position and plane of cell divi and orientation of the daughter cells. ision influences the size Changes in cell shape and size drive many folding and buckling movements, such as those occurring during gastrulation and the formation of the neural tube. Cell size and shape can change due to reorganization of the cytoskeleton, or the uptake of water or lipids. Cell fusion generates a multinucleate syncytium. This is important in the morphogenesis of developing muscle tissue and that of the trophectoderm during implantation in mammals. Programmed cell death by apoptosis in development is responsible for creating cavities (such as the proamniotic cavity in mouse development) and gaps (e.g, between the digits in vertebrate limb development). Extensive cell death also occurs in the developing nervous system as neurons compete for innervation targets. ‘Tissue reorganization (transitions between epithelial and mesenchyme cells, delamination, intercalation) occur through changes in the pattern of cell adhesion molecules. The expression of different classes of cell adhesion molecules helps to keep like cells together in tissues, and maintain boundaries between different tissues. The extracellular matrix is a network of macromolecules secreted by cells into their local environment. Interactions between cells and the matrix can maintain epithelial sheets, provide a substrate for migration, and induce differentiation. The matrix is also the predominant component of some tissues, such as bone and cartilage. ‘The cell division cycle (C4) The cytoskeleton, cell-adhesion and the extracellular matrix (C5)AT ~ Morphogenesis 39 Mechanisms of morphogenesis Cell division ‘The developing embryo is dynamic, and forms all manner of shapes and struc- tures. This is achieved through various different types of cell behavior. The term morphogenesis (creation of form) is used to describe how embryonic structures arise. Without morphogenesis, the embryo would never progress beyond a simple ball of cells, and dynamic processes such as gastrulation, embryonic folding and organogenesis would not be possible. A number of morphogenetic mechanisms are listed in Table 1, together with examples from different develop- mental systems. The importance of each of these processes is discussed below. Table 1. Morphogenetic processes in development and examples from model develop- mental systems Process Examples Differential rates of cell proliferation Selective outgrowth of vertebrate limb buds by proliferation of cells in the progress zone. Growth of shoots and roots in mature plants by prolifera- tion of meristems. Alternative positioning and/or Different embryonic cleavage patterns in animals. orientation of mitotic spindle Stereotyped cell divisions in nematodes and Arabidopsis roots. Switch from anticlinal to periclinal divisions in leaf development Change of cell size Cell expansion during leaf development in Arabidopsis Change of cell shape Change from columnar to wedge-shaped cells during neural tube closure in birds and mammals Cell fusion, Formation of trophoblast and myotubes in mammals, Cell death Separation of digits in vertebrate limb bud Selection of functional synapses in the mammalian nervous system. Gain of cell-cell adhesion Condensation of cartilage mesenchyme in vertebrate limb bud, Loss of cell-cell adhesion Delamination of cells from epibiast during gastru- lation in mammals. Celi-matrix interaction Migration of neural crest cells and germ cells, ‘Axon migration. Loss of cellematrix adhesion Delamination of cells from basal layer of the epidermis. Cell division is important for morphogenesis in two ways. Firstly, the rate of cell division contributes to the differential growth of parts of the embryo. This can be seen in the vertebrate embryo for example, in the selective growth of the limb bud rather than adjacent regions of the lateral plate mesoderm. Such growth results from the secretion of growth factors firstly by the mesoderm itself, and then by the apical ectodermal ridge overlying the limb bud. The growth factors diffuse only a short distance, resulting in a small but rapidly dividing growth zone at the tip of the limb bud, from which all the skeletal and connective tissue elements of the limb derive (Topic MD.