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During development, gene activation is stringently regulated to restrict expression only to the correct cell type and correct developmental stage. Here, we present mechanistic evidence that suggests DNA methylation contributes to this regulation by suppressing premature gene activation. Using the mouse Myogenin promoter as an example of the weak CpG island class of promoters, we find that it is initially methylated but becomes demethylated as development proceeds. Full hypersensitive site formation of the Myogenin promoter requires both the MEF2 and SIX binding sites, but binding to only one site can trigger the partial chromatin opening of the nonmethylated promoter. DNA methylation markedly decreases hypersensitive site formation that now occurs at a detectable level only when binding to both MEF2 and SIX binding sites is possible. This suggests that the probability of activating the methylated promoter is low until two of the factors are coexpressed within the same cell. Consistent with this, the single-cell analysis of developing somites shows that the coexpression of MEF2A and SIX1, which bind the MEF2 and SIX sites, correlates with the fraction of cells that demethylate the Myogenin promoter. Taken together, these studies imply that DNA methylation helps to prevent inappropriate gene activation until sufficient activating factors are coexpressed.

Within the mammalian genome, there are many multimember gene families that encode membrane proteins with extracellular leucine rich repeats which are thought to act as cell adhesion or signalling molecules. We previously showed that the members of the NLRR gene family are expressed in a developmentally restricted manner in the mouse with NLRR-1 being expressed in the developing myotome. The FLRT gene family shows a similar genomic layout and predicted protein secondary structure to the NLRRs so we analysed expression of the three FLRT genes during mouse development. FLRTs are glycosylated membrane proteins expressed at the cell surface which localise in a homophilic manner to cell–cell contacts expressing the focal adhesion marker vinculin. Each member of the FLRT family has a distinct, highly regulated expression pattern, as was seen for the NLRR family. FLRT3 has a provocative expression pattern during somite development being expressed in regions of the somite where muscle precursor cells migrate from the dermomyotome and move into the myotome, and later in myotomal precursors destined to migrate towards their final destination, for example, those that form the ventral body wall. FLRT3 is also expressed at the midbrain/hindbrain boundary and in the apical ectodermal ridge, regions where FGF signalling is known to be important, suggesting that the role for FLRT3 in FGF signalling identified in Xenopus is conserved in mammals. FLRT1 is expressed at brain compartmental boundaries and FLRT2 is expressed in a subset of the sclerotome, adjacent to the region that forms the syndetome, suggesting that interaction with FGF signalling may be a general property of FLRT proteins. We confirmed this by showing that all FLRTs can interact with FGFR1 and FLRTs can be induced by the activation of FGF signalling by FGF-2. We conclude that FLRT proteins act as regulators of FGF signalling, being induced by the signal and then able to interact with the signalling receptor, in many tissues during mouse embryogenesis. This process may, in part, be dependent on homophilic intercellular interactions between FLRT molecules.

During vertebrate embryogenesis, the somites form by segmentation of the trunk mesoderm, lateral to the neural tube, in an anterior to posterior direction. Analysis of differential gene expression during somitogenesis has been problematic due to the limited amount of tissue available from early mouse embryos. To circumvent these problems, we developed a modified differential display PCR technique that is highly sensitive and yields products that can be used directly as in situ hybridisation probes. Using this technique, we isolated NLRR-1 as a gene expressed in the myotome of developing somites but not in the presomitic mesoderm. Detailed expression analysis showed that this gene was expressed in the skeletal muscle precursors of the myotome, branchial arches and limbs as well as in the developing nervous system. Somitic expression occurs in the earliest myoblasts that originate from the dorsal lip in a pattern reminiscent of the muscle determination gene Myf5, but not at the ventral lip, indicating that NLRR-1 is expressed in a subset of myotome cells. The NLRR genes comprise a three-gene family encoding glycosylated transmembrane proteins with external leucine-rich repeats, a fibronectin domain, an immunoglobulin domain and short intracellular tails capable of mediating protein–protein interaction. Analysis of NLRR-3 expression revealed regulated expression in the neural system in developing ganglia and motor neurons. NLRR-2 expression appears to be predominately confined to the adult. The regulated embryonic expression and cellular location of these proteins suggest important roles during mouse development in the control of cell adhesion, movement or signalling.

The myogenic regulatory factor Myf5 is integral to the initiation and control of skeletal muscle formation. In adult muscle, Myf5 is expressed in satellite cells, stem cells of mature muscle, but not in the myonuclei that sustain the myofibre. Using the Myf5nlacZ/+ mouse, we now show that Myf5 is also constitutively expressed in muscle spindles-stretch-sensitive mechanoreceptors, while muscle denervation induces extensive reactivation of the Myf5 gene in myonuclei. To identify the elements involved in the regulation of Myf5 in adult muscle, we analysed reporter gene expression in a transgenic bacterial artificial chromosome (BAC) deletion series of the Mrf4/Myf5 locus. A BAC carrying 140 kb upstream of the Myf5 transcription start site was sufficient to drive all aspects of Myf5 expression in adult muscle. In contrast, BACs carrying 88 and 59 kb upstream were unable to drive consistent expression in satellite cells, although expression in muscle spindles and reactivation of the locus in myonuclei were retained. Therefore, as during development, multiple enhancers are required to generate the full expression pattern of Myf5 in the adult. Together, these observations show that elements controlling adult Myf5 expression are genetically separable and possibly distinct from those that control Myf5 during development. These studies are a first step towards identifying cognate transcription factors involved in muscle stem cell regulation.

Hox genes are key determinants of anteroposterior patterning of animal embryos, and spatially restricted expression of these genes is crucial to this function. In this study, we demonstrate that expression of Hoxb4 in the paraxial mesoderm of the mouse embryo is transcriptionally regulated in several distinct phases, and that multiple regulatory elements interact to maintain the complete expression domain throughout embryonic development. An enhancer located within the intron of the gene (region C) is sufficient for appropriate temporal activation of expression and the establishment of the correct anterior boundary in the paraxial mesoderm (somite 6/7). However, the Hoxb4 promoter is required to maintain this expression beyond 8.5 dpc. In addition, sequences within the 3′ untranslated region (region B)are necessary specifically to maintain expression in somite 7 from 9.0 dpc onwards. Neither the promoter nor region B can direct somitic expression independently, indicating that the interaction of regulatory elements is crucial for the maintenance of the paraxial mesoderm domain of Hoxb4expression. We further report that the domain of Hoxb4 expression is restricted by regulating transcript stability in the paraxial mesoderm and by selective translation and/or degradation of protein in the neural tube. Moreover, the absence of Hoxb4 3′-untranslated sequences from transgene transcripts leads to inappropriate expression of some Hoxb4transgenes in posterior somites, indicating that there are sequences within region B that are important for both transcriptional and post-transcriptional regulation.

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.

The development of skeletal muscle in vertebrate embryos is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf5, Myogenin, MRF4 and MyoD. In the mouse embryo, Myf5 is the first of these factors to be expressed and mutational analyses suggest that this protein acts early in the process of commitment to the skeletal muscle fate. We have therefore analysed the regulation of Myf5 gene expression using transgenic technology and find that its control is markedly different from that of the other two myogenic regulatory factor genes previously analysed, Myogenin and MyoD. We show that Myf5 is regulated through a number of distinct and discrete enhancers, dispersed throughout 14 kb spanning the MRF4/Myf5 locus, each of which drives reporter gene expression in a particular subset of skeletal muscle precursors. This region includes four separate enhancers controlling expression in the epaxial muscle precursors of the body, some hypaxial precursors of the body, some facial muscles and the central nervous system. These elements separately or together are unable to drive expression in the cells that migrate to the limb buds and in some other muscle subsets and to correctly maintain expression at late times. We suggest that this complex mechanism of control has evolved because different inductive signals operate in each population of muscle precursors and thus distinct enhancers, and cognate transcription factors, are required to interpret them.

There is increasing evidence that retinoic acid (RA), a vitamin A metabolite, plays a role in the development of the nervous system. Here we specifically test this notion by examining the effect of RA on neurite outgrowth from explanted segments of the axolotl spinal cord. We show that there is a threshold concentration in the region of 0.1-1 nM above which neurite outgrowth is stimulated 4-5 fold. Retinol, by contrast, only stimulated the migration of glial cells from the explants. Using HPLC we demonstrate that RA and retinol are present endogenously in the axolotl spinal cord. In addition, we have identified by immunocytochemistry with antipeptide antibodies the cells of the spinal cord that contain the binding proteins for RA (cellular RA-binding protein; CRABP) and retinol (cellular retinol-binding protein; CRBP). CRABP is found in the axons and CRBP is found in the ependyma and glial cells. These results provide strong evidence for a role for RA in the developing nervous system, and we propose a specific hypothesis involving CRBP, CRABP, retinol, and RA in the control of axon outgrowth in the spinal cord.

Pattern formation in both the developing and the regenerating vertebrate limb can be understood in terms of the assignment of positional values to cells. The positional value may then control the selection of a course of cytodifferentiation as, say, muscle or cartilage. It is proposed that the proximodistal coordinate of positional value is extablished by a mechanism based on autonomous change with time in a 'progress zone' at the tip of the limb bud. The anteroposterior coordinate seems to be specified by a positional signal from an orgainzing region. Only cells in the progress zone respond to this signal. The change of character in the progress zone may be linked to cell division. It seems that each skeletal rudiment may initially be the same length, corresponding to the amount of tissue that leaves the progress zone during one cell cycle. The lengths on the skeletal elements are already specified shortly after exit from the progress zone, and are capable of very little regulation. The positional value may determine the pregramme of later growth. The relevance of the progress zone model to epimorphic regulation, and amphibian limb regeneration in particular, is discussed.

Myf5, the skeletal muscle determination gene, is first expressed in the dorso-medial aspect of the somite under the control of an element we have called the early epaxial enhancer. It has subsequently been reported that this enhancer is a direct target of Shh signaling mediated by Gli transcription factors (Gustafsson et al. 2002). We here demonstrate that activation of Myf5 expression depends on neither Shh function nor an intact Gli binding site, although the Gli site is necessary for continuation of expression. We suggest that the discrepancy is due to the existence of specific interactions between the enhancer and the Myf5 promoter.

Myf5 is the first myogenic regulatory factor to be expressed in the mouse embryo and it determines the entry of cells into the skeletal muscle programme. A region situated between -58 kb and -48 kb from the gene directs Myf5 transcription at sites where muscles will form. We now show that this region consists of a number of distinct regulatory elements that specifically target sites of myogenesis in the somite, limbs and hypoglossal cord, and also sites of Myf5 transcription in the central nervous system. Deletion of these sequences in the context of the locus shows that elements within the region are essential, and also reveals the combinatorial complexity of the transcriptional regulation of Myf5. Both within the -58 kb to -48 kb region and elsewhere in the locus, multiple sequences are present that direct transcription in subdomains of a single site during development, thus revealing distinct phases of myogenesis when subpopulations of progenitor cells enter the programme of skeletal muscle differentiation., This work in M.B.'s laboratory was supported by the Pasteur Institute and the CNRS and by grants from the ACI Integrative Biology Programme of the MJER, the AFM and the European Community (QLK3-CT-99/02). J.H. benefited from fellowships from ARC and the AFM, L.B. from funding from the MJER, and T.C. from fellowships from NIH and the AFM. The work in P.R.'s laboratory was supported by a grant from The Institute of Cancer Research.

Vertebrate myogenesis is controlled by four transcription factors known as the myogenic regulatory factors (MRFs): Myf5, Mrf4, myogenin and MyoD. During mouse development Myf5 is the first MRF to be expressed and it acts by integrating multiple developmental signals to initiate myogenesis. Numerous discrete regulatory elements are involved in the activation and maintenance of Myf5 gene expression in the various muscle precursor populations, reflecting the diversity of the signals that control myogenesis. Here we focus on the enhancer that recapitulates the first phase of Myf5 expression in the epaxial domain of the somite, in order to identify the subset of cells that first transcribes the gene and therefore gain insight into molecular, cellular and anatomical facets of early myogenesis. Deletion of this enhancer from a YAC reporter construct that recapitulates the Myf5 expression pattern demonstrates that this regulatory element is necessary for expression in the early epaxial somite but in no other site of myogenesis. Importantly, Myf5 is subsequently expressed in the epaxial myotome under the control of other elements located far upstream of the gene. Our data suggest that the inductive signals that control Myf5 expression switch rapidly from those that impinge on the early epaxial enhancer to those that impinge on the other enhancers that act later in the epaxial somite, indicating that there are significant changes in either the signalling environment or the responsiveness of the cells along the rostrocaudal axis. We propose that the first phase of Myf5 epaxial expression, driven by the early epaxial enhancer in the dermomyotome, is necessary for early myotome formation, while the subsequent phases are associated with cytodifferentiation within the myotome.

The development of skeletal muscle in vertebrate embryos is controlled by a transcriptional cascade involving the four myogenic regulatory factors. In the somites of the mouse embryo the order of expression is thought to be Myf5, Myogenin, Mrf4 and MyoD. We have re-examined the expression pattern of Mrf4 and show that in the hypaxial domain of thoracic somites (the somitic bud) Mrf4 expression precedes or is contemporaneous with that of Myf5, suggesting that this transcription factor plays a hitherto unsuspected role in myogenesis.

Publisher Summary This chapter discusses the biochemical mechanisms involved in the regulation of transcription during the development of the mouse embryo. General approach for this study involves taking genes known to act at a fairly high level in a particular genetic pathway and then to try to understand how their transcription is controlled using the power of transgenic mouse technology. In each case the objective is to identify of each of the transcription factors that control the chosen regulatory gene. It is reported that segmental identity in the vertebrate embryo is controlled by the four clusters of Hox genes. Therefore, the chapter analyzes the regulation of one such gene, Hoxb-4. One of the earliest transcriptional responses in the epithelial somite is the activation of the cascade of basic helix-loop-helix (bHLH) transcription factors that together regulate the commitment of skeletal myoblasts and their subsequent differentiation. Although there is a considerable knowledge of genes that function during somitogenesis, it is clear that many more such genes remain unknown and one has therefore sought to develop techniques that may allow the discovery of novel genes that act during defined aspects of the process.

We describe here two aspects of our work on retinoic acid (RA) and pattern formation in the regenerating limb. The first concerns the presence of endogenous RA in the regenerating limb and the second concerns experiments on the role of cellular retinoic acid-binding protein (CRABP) in the process of pattern respecification.

Ten years ago research into limb development and regeneration had produced a wealth of material describing interesting biological phenomena. These included the identification of signalling and responding regions, descriptions of the pattern of changes of cell differentiation and cell division and the ability to manipulate the embryo so as to modify these patterns almost at will. The intellectual force behind much of the research lay in a wealth of theoretical papers discussing possible mechanisms. These in turn stimulated new waves of experiments. What the system lacked was any clear evidence supporting a particular molecular mechanism. However within a very few years the situation has been transformed with the appearance of three distinct lines of enquiry that have converged to produce a key that may unlock the secrets of development.

The vertebrate limb bud develops as an autonomous embryonic field. The pattern of differentiation within the bud is at least in part controlled by discrete organising regions. While the interactions involved are probably complex the system can be simply modelled as three independent linear orthogonal axes. For some time the fashionable, or to use the correct molecular biology term, the sexy study, has been the antero-posterior (AP) axis and its discrete organisor, the Zone of Polarising Activity (ZPA).

Retinoic acid has been used as a tool both by embryologists studying the spatial organization of cells in the embryo and by molecular biologists studying the control of gene expression in the nucleus. Embryologists have shown that retinoic acid can modify the pattern of cell differentiation so as to duplicate complete parts of the embryo in a well-organized way; molecular biologists have shown that retinoic acid can act as the switch starting the sequence of differential gene expression that results in cell differentiation. In the past year these two approaches have converged so that there now seems a real possibility that we may soon for the first time understand how a particular vertebrate development system works.

Sections of the proximal-distal axis were removed so as to produce wings lacking entire skeletal levels. The innervation of the resulting limb from the appropriate spinal nerves was tested by electrophysiological recording, gross dissection and serial sections. The nerves form repeatable and ordered patterns in the modified limbs which can be readily related to the normal pattern. In general the segmental nerves only innervate their appropriate territory irrespective of its location; the route followed by the nerves is characteristic for the skeletal level they traverse. If a target is missing then so is the normal nerve branch to that region.

Tissue from the posterior margin of the developing limb bud, when grafted to the anterior margin, evokes the formation of a mirror-image limb duplication from the host tissue. We present maps of the spatial and temporal distribution of this signalling activity in the chick wing bud based on a bioassay that provides a quantitative measure of the completeness of the additional structures (the strength of activity index). Activity is first detected prior to the initial appearance of the limb primordium as early as Hamburger & Hamilton stage 14. It reaches a maximum during early outgrowth of the bud at stages 19 to 25. It then declines as the limb starts to differentiate into its final morphological pattern. The design of the experiment provides serendipitous data showing that two operators can consistently perform grafts with high reproducibility between them while variability between embryos is somewhat higher. The maps of activity are of particular practical value in precisely defining for the experimental embryologist and molecular biologist those positions and stages at which peak signalling activity resides.

Transient stimulation of cell division within chick wingbud mesenchyme is associated with the reorganization of limb pattern that follows insertion of a small graft from the posterior zone of limb polarizing activity into the anterior border of a host bud. This increase is initiated as an enhanced rate of entry to S phase, correlated with a decrease in packing density among mesenchyme cells, through much of the limb rudiment a few hours after the operations.

The wing-bud shows zero regulation following removal of a whole slice of the proximaldistal axis from stage 22 or later. Prior to this it is possible that it may show some regulation. Some of the apparent regulation may be explained by the way in which the limb grows. The bud never shows perfect size regulation or morphallaxis. The proximal-distal axis of the bud between shoulder and wrist expands uniformly from stage 20.

Quantitative experimental evidence is presented for the progress zone theory of limb development. The theory, here formulated mathematically, states that the parts of the limb are specified in proximo-distal succession by an autonomous timing mechanism operating in a ‘progress zone’ of undifferentiated growing mesenchyme under the influence of the apical ectodermal ridge. By the exchange of distal tips between young and old wing-buds, it is shown that there are no long-range morphogenetic signals from proximal to distal tissue. The width of the progress zone is calculated, and it is found autoradiographically that practically all its cells are dividing.

Quail grafts of Hensen’s node were examined for their potential to induce somites in chick blastoderms. The origin of the structures induced depended on the distance of the graft from the host’s midline. Nodes placed at the margin of the area pellucida resulted in structures differentiated from the cells of the graft, whereas medially the graft organized host cells to form rows of somites. The results are discussed in terms of competence of graft and host mesenchyme and a positional signal from the node.

The effect of removal of the apical ectodermal ridge from the early chick limb-bud is re-examined using a new quantitative method of analysis of results. The concept of the proximo-distal sequence of laying down of parts is confirmed and evidence is presented that this proceeds as a continuous process, there being a gradual change in the level specified from one cell to another at a more distal level. The results are then interpreted in terms of the ‘progress zone’ model to show that they are both consistent with the model and that they provide an assay for one of its parameters, the rate of change of positional value with time at the tip.

SYNOPSIS. The proposition that pattern formation across the antero-posterior axis of the developing chick limb is controlled by a special signalling region has prompted many experiments. No result is inconsistent with the basic hypothesis, and much evidence supports the notion of a graded signal from the zone of polarizing activity. We describe the location in space and time, the size, the graded nature of the signal and the distance over which it acts; but despite increasing interest the molecular mechanism remains unknown. A major change in our understanding of the interaction between the polarizing region and the responding limb field is that growth as well as pattern is involved.

The Royal Society devoted the 25th and 26th of March to a discussion of theories of biological pattern formation 1 . The meeting reviewed the work of the last decade in a style that should make it intelligible to the general reader, and it provided encouragement that the next decade will be equally exciting. I review here not the individual papers, but the general theme. So many different competing ideas exist in the field that it is often difficult to arrive at any generalization or even to resolve the conflicting nomenclature.

SYNOPSIS. There is a consistent repeatable pattern of innervation of the vertebrate limb by sensory and motor axons from the spinal cord. We have been looking at the factors which control the generation of this pattern in the developing chick limb. The gross pattern of nerves in the limb is controlled by the limb tissue and arises as a result of the axon's response to its local environment. Each segmental root innervates a characteristic territory in the limb. Each muscle is innervated by a characteristically located motoneurone pool in the cord. Three models explaining the generation of these patterns are presented: active selection, selective cell death, and passive deployment. A review of recent experiments leads us to conclude that there is evidence for both passive deployment and local, short range, active selection.

The trajectories of motor axons innervating chick wings reversed about the DV or AP axis before axon invasion were analysed after retrograde filling by HRP injection into biceps or triceps muscles. Chick-quail chimaeras showed that the plane of reversal for flank operations was proximal to the confluence of the 14th, 15th, and 16th spinal roots as they form the plexus. The shoulder reversal plane was distal to the plexus. In dorsoventral (DV) reversed wings at both shoulder or flank level, the motor axons do not alter their course as they enter the graft. They therefore innervate by passive deployment any target that they encounter. In anteroposterior (AP) reversed wings at both shoulder or flank level, the motor axons clearly corrected their position in the nerve tract after entering the graft and innervated appropriate targets. The innervation of appropriate targets in AP shoulder reversals shows that axons are sensitive to AP mismatch distal to the plexus. Since axons were displaced similar distances from their normal routes in flank DV and AP reversals, the difference in behaviour suggests that they respond to mismatch in the AP but not the DV axis.

CHIMAERAS (or “allophenic individuals”) let us see the pedigree of cells. For instance, a blastocyst compounded from a black-coated-mouse embryo and a white-coated-mouse embryo may develop into a striped or piebald adult mouse1. In other adult tissues, other markers (such as isozymes) reveal a similar conglomerate of two cell types2. Naturally occurring mosaics, such as those which arise according to the Lyon hypothesis through random X-inactivation in females, provide further examples3,4. In every case, the chimaeric pattern records something of the developmental history of the individual. Indeed, it has been claimed that the assortment of cell types in the chimaeric differentiated tissue provides some crucial evidence about the mechanisms of differentiation and morphogenesis. In particular, Mintz1 and Tettenborn et al.4 have sought to infer the initial numbers of specific tissue progenitors from observations of how often, in mosaic animals, those tissues are not mosaic, but homogeneous. We shall analyse this type of inference, and argue that the conclusions to which it properly leads do not have quite the significance that has been suggested.

Retinoic acid may be the natural morphogen used to generate digit pattern in the chick limb bud. It has been proposed that retinoic acid acts by binding to a cellular retinoic acid-binding protein (CRABP) and then entering the nucleus to alter the pattern of gene activity. High-affinity receptors that bind both retinoic acid and DNA and are analogous to the steroid receptors have been identified. But the concentration of endogenous retinoic acid in the limb and the binding coefficient of the nuclear receptors indicate that they are saturated throughout the limb. Here we investigate the CRABP distribution in the developing chick limb bud. We find CRABP in the area of intense morphogenetic activity at the tip, with a differential distribution across the anteroposterior axis, the high point being at the anterior margin. Retinoic acid also forms a concentration gradient across the limb bud, but is highest on the posterior side. We propose that CRABP could be reducing the effective concentration of retinoic acid reaching the nucleus to a level appropriate for the differential regulation of gene transcription, providing a spatially modulated morphogenetic gradient of information for digit formation.

THE chick wing is innervated by adjacent spinal roots which meet at the brachial plexus and then redistribute their axons to the peripheral nerves. Both the anatomical arrangement of nerves within the wing and the innervation territories supplied by each root are constant among animals of the same strain1–3. We describe here the pathways taken by the axons from each of the main roots to their particular territories in the normal wing, and also in wings which have dorso-ventral and antero-posterior axes reversed relative to the host axes. Our observations suggest that the characteristic innervation territories of the segmental roots may arise from two basic rules: that axons travel in parallel over long distances without crossing, and that the branching pattern of nerves within the limb is determined by the local environment3,4.

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.

Removal of the apical ectodermal ridge causes a reduction in the rate of outgrowth of the wing-bud and the loss of distal parts. More specifically it causes a short-term increase in cell density and cell death and a decrease in the rate of cell proliferation. The evidence supports the hypothesis of density-dependent control of cell division and suggests that there may also be a mechanism regulating skeletal length at the time of differentiation. An informal model is presented to explain the observations.

Local application of retinoic acid to the chick limb bud produces effects that are dose and/or stage dependent. Low doses and/or old stages tend to give normal limbs or perhaps one or two supernumerary digits of a more anterior character. Medium doses and/or intermediate stages tend to give full mirror-image supernumeraries with two or even three extra digits including particularly digits of a posterior character. High doses and/or early stages give limbs in which supernumerary digits fail to form or are lost, and in which even host skeletal elements are missing or reduced. The effects are graded over the full dose and/or stage range. Various explanations are discussed in the context of the current hypotheses of limb development. We conclude that one should not necessarily interpret the results as evidence that retinoids normally play a part in the control of development or regeneration.

The interpretation of positional information can provide the basis for pattern formation in limb morphogenesis. The gradient in positional information along the antero-posterior axis, which is specified with respect to a localised boundary region, can be modified by grafting this region to successive positions along the axis. The pattern of digits obtained is consistent with a model based on diffusion of a labile morphogen and is thus similar to models proposed for the development of pattern in invertebrates.

The experiments examine the extent of reduplication of skeletal parts across the anteroposterior axis, following the transplantation of a zone of polarizing activity (ZPA) to the anterior margin of the limb-bud at successively later stages. Previous studies have suggested that the function of the apical ectodermal ridge (AER) is to maintain cells in a special region at the distal tip (the progress zone) labile, with respect to their positional value along the proximo-distal axis. Similarly, the results of these experiments demonstrate that cells in the progress zone are able to change their antero-posterior positional value under the influence of the grafted ZPA, while cells at more proximal levels remain unaffected. In turn, the ZPA may effect the activity of the AER and hence the progress zone.

Differentiation of the wing skeleton is clearly visible in whole mounts from stage 24. It proceeds in proximo-distal and postero-anterior sequence. It is possible to map the part of the limb giving rise to each skeletal element as it appears and subsequently. This enables one to estimate: (a) a rate of elongation curve for each segmental level, (b) the intrinsic rate of change of elongation of the cartilage, (c) a definitive extrapolation back to classical presumptive fate maps, (d) the normal range of variation in the proportion of the limb occupied by the three main segmental levels (stylopod, autopod, zeugopod).

“It is not birth, marriage or death, but gastrulation which is truly the most important time in your life.” Lewis Wolpert quoted in Slack, 1983. While one can hardly mistake childbirth, and while half the world has been known to stop for a wedding, gastrulation is neither obvious nor dramatic for the onlooker. The real show stopping events are the processes of neurulation and segmentation which produce recognisable living organisms from a featureless surface (Figure 1).

This paper examines the hypothesis that the developing chick limb bud has mechanisms for regulating the control of growth, size and pattern. The tests included: surgical removal of selected parts of the limb field, X-irradiation, temperature shock and the manipulation of known limb organizer regions (removal of the apical ectodermal ridge, or the addition of an extra zone of polarizing activity). The results strongly support the idea that there are regulatory mechanisms controlling both the pattern and the size of the limb and suggest that they involve regulation of the growth rate via control of cell division throughout the embryonic period. Possible mechanisms are discussed.

Publisher Summary During embryogenesis, a single fertilized egg cell develops into a complex organism consisting of many cell types arranged in a reproducible pattern. The development of spatial organization of the differentiated cells is called pattern formation. Many different processes occur, including cell movements, changes in cell division, appearances of overt differences between cells, and restriction of developmental potentialities. The operational unit of pattern formation is the embryonic field, which is a region in which cells of identical or nearly so characteristics, autonomously develop into a pattern of different cell types. This chapter discusses several mechanisms of pattern formation, the concept of positional information, and the way each cell knows its appropriate behavior within the entire organism. The limb is a well-studied model system for understanding the development of pattern. Historically, the three-dimensional structure of the limb has been anatomically partitioned into three orthogonal coordinate axes—dorsoventral, anteroposterior, and proximodistal.