Fate maps old and new.

Fate mapping was once the province of classical experimental embryologists. Now a battery of new and sophisticated methods can be used to trace where cells go and what they do in embryos. Here we use examples from gastrulating fish and amphibian embryos and from the chick limb bud and central nervous system to show how this information has contributed to our understanding of developmental processes. This knowledge will become increasingly important in interpreting the complex patterns of gene expression that are being discovered during development, as well as in understanding the effects of genetic manipulations and in directing experimental interventions.

Patterning systems--from one end of the limb to the other.

A combination of embryology and gene identification has led us to the current view of vertebrate limb development, in which a series of three interlocking patterning systems operate sequentially over time. This review describes current understanding of these regulatory mechanisms and how they form a framework for future analysis of limb patterning.

Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects.

All-trans-retinoic acid causes striking digit pattern changes when it is continuously released from a bead implanted in the anterior margin of an early chick wing bud. In addition to the normal set of digits (234), extra digits form in a mirror-symmetrical arrangement, creating digit patterns such as a 432234. These retinoic acid-induced pattern duplications closely mimic those found after grafts of polarizing region cells to the same positions with regard to dose-response, timing, and positional effects. To elucidate the mechanism by which retinoic acid induces these pattern duplications, we have studied the temporal and spatial distribution of all-trans-retinoic acid and its potent analogue TTNPB in these limb buds. We find that the induction process is biphasic: there is an 8-h lag phase followed by a 6-h duplication phase, during which additional digits are irreversibly specified in the sequence digit 2, digit 3, digit 4. On average, formation of each digit seems to require between 1 and 2 h. The tissue concentrations, metabolic pattern, and spatial distribution of all-trans-retinoic acid and TTNPB in the limb rapidly reach a steady state, in which the continuous release of the retinoid is balanced by loss from metabolism and blood circulation. Pulse-chase experiments reveal that the half-time of clearance from the bud is 20 min for all-trans-retinoic acid and 80 min for TTNPB. Manipulations that change the experimentally induced steep concentration gradient of TTNPB suggest that a graded distribution of retinoid concentrations across the limb is required during the duplication phase to induce changes in the digit pattern. The extensive similarities between results obtained with retinoids and with polarizing region grafts raise the possibility that retinoic acid serves as a natural "morphogen" in the limb.

Fibroblast growth factor 4 directs gap junction expression in the mesenchyme of the vertebrate limb Bud.

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. Here we address the underlying cellular mechanisms. We show in the intact bud that connexin 43 (Cx43) and Cx32 gap junctions are at higher density between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. We find that FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. We show that posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. We conclude that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected.

Vertebrate limb development.

The recent identification of Wnt-7a as a signalling molecule in dorsal/ventral patterning means that we now have a known signal for control of each of the three limb axes. Fibroblast growth factors can allow proximal/distal patterning and Sonic hedgehog gene expression signals anterior/posterior patterning. Networks of these signals not only coordinate cell responses, but also mutually maintain each other. A positive feedback loop is established which coordinates expression of Sonic hedgehog in mesenchyme cells of the polarizing region and Fgf-4 expression in overlying apical ridge ectoderm. Wnt-7a expression in dorsal ectoderm also influences Sonic hedgehog expression in the polarizing region. Initiation of development of a complete limb can be achieved with just one signal, a growth factor.

Epithelial cell movements and interactions in limb, neural crest and vasculature.

Formation of the thickened apical ectodermal ridge of developing vertebrate limbs appears to be a complex process. Direct connections to molecular controls of cell migratory machinery have been shown for first time in neural crest migration. New unsuspected roles are emerging for ephrin ligand/Eph receptor signalling in vascular morphogenesis.

Vertebrate limb development--the early stages in chick and mouse.

More news this year about FGFs and their roles in vertebrate limb initiation; Wnt signalling is shown for the first time to be another component of the signalling cascade involved in early limb formation. Ectodermal compartments that control apical ridge formation were previously described in chick embryos and are now shown to exist in mouse embryos; Engrailed1 is expressed in the ventral ectodermal compartment but experiments in both chick and mouse show that it is not responsible for compartment specification.

Limbs: a model for pattern formation within the vertebrate body plan.

Giant strides have been made in identifying the molecular basis of limb development. The four main phases are initiation of the limb bud, specification of limb pattern, differentiation of tissues and shaping of the limb, and growth of the miniature limb to the adult size. We will focus on the exciting advances that have been made in initiation and specification of limb pattern. The limb is a model system and the same sets of molecules are used at different times and places in vertebrate embryos. There is also remarkable conservation of the molecular mechanisms of limb development in insects and vertebrates.

The chicken as a model for embryonic development.

The traditional strength of chicken embryos for studying development is that they are readily manipulated. This has led to some major discoveries in developmental biology such as the demonstration that the neural crest gives rise to almost the entire peripheral nervous system and the identification of signalling centres that specify the pattern of structures in the central nervous system and limb. More recently with the burgeoning discovery of developmentally important genes, chicken embryos have provided useful models for testing function. Uncovering the molecular basis of development provides direct links with clinical genetics. In addition, since many genes that have crucial roles in development are also expressed in tumours, basic research on chickens has implications for understanding human health and disease. Now that the chicken genome has been sequenced and genomic resources for chicken are becoming increasingly available, this opens up opportunities for combining these new technologies with the manipulability of chicken embryos and also exploiting comparative genomics.

The contribution of chicken embryology to the understanding of vertebrate limb development.

The chicken is an excellent model organism for studying vertebrate limb development, mainly because of the ease of manipulating the developing limb in vivo. Classical chicken embryology has provided fate maps and elucidated the cell-cell interactions that specify limb pattern. The first defined chemical that can mimic one of these interactions was discovered by experiments on developing chick limbs and, over the last 15 years or so, the role of an increasing number of developmentally important genes has been uncovered. The principles that underlie limb development in chickens are applicable to other vertebrates and there are growing links with clinical genetics. The sequence of the chicken genome, together with other recently assembled chicken genomic resources, will present new opportunities for exploiting the ease of manipulating the limb.

Expression patterns of Notch1, Serrate1, Serrate2 and Delta1 in tissues of the developing chick limb.

Signalling via the receptor Notch, delivered by the ligands Delta and Serrate, plays a key role in many cell fate decisions in both Drosophila and vertebrate development (for review seeArtavanis-Tsakonas, S., Matsuno, K. and Fortini, M.E., 1995. Notch signalling. Science 268, 225-232; Lewis, J., 1996. Neurogenic genes and vertebrate neurogenesis. Curr. Opin. Neurobiol. 6, 3-10; Blair, S.S., 1997. Limb development: marginal fringe benefits. Curr Biol. 7, 686-690; Irvine, K.D. and Vogt, T.F., 1997. Dorsal-ventral signaling in limb development. Curr. Opin. Cell Biol. 9, 867-876). Recently vertebrate homologues of Notch (Notch1; Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J., 1996. A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogeneis. Dev. Biol. 174, 233-247) and Serrate (Serrate1 and 2; Myat, A., Henrique, D., Ish-Horowicz, D. and Lewis, J., 1996. A chick homologue of Serrate and its relationship with Notch and Delta homologues during central neurogeneis. Dev. Biol. 174, 233-247; Hayashi, H., Mochii, M., Kodama, R., Hamada, Y., Mizuno, N., Eguchi, G. and Tachi, C., 1996. Isolation of a novel chick homolog of Serrate and its coexpression with Notch-1 in chick development. Int. J. Dev. Biol. 40, 1089-96; Laufer, E., Dahn, R., Orozco, O.E., Yeo, C.Y., Pisenti, J., Henrique, D., Abbott, U., Fallon, J.F. and Tabin, C., 1996. Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature 386, 366-373; Rodriguez-Esteban, C., Schwabe, J.W., De La Pena, J., Foys, B., Eshelman, B. and Izpisua-Belmonte, J.C., 1997. Radical fringe positions the apical ectodermal ridge at the dorsoventral boundary of the vertebrate limb. Nature 386, 360-366) were shown to be expressed in early chick limb mesenchyme and apical ridge. However, later expression patterns of these genes and of Delta 1 (Henrique, D. , Adam, J., Myat, A., Chitnis, A., Lewis, J. and Ish-Horowicz, D., 1995. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787-790) in vertebrate limbs have not been documented. We have used whole mount in-situ hybridization to document expression patterns of Notch1, Serrate1, Serrate2 and Delta1 within the mesenchyme of the developing chick limb up to stage 31 of development. We show these genes are expressed, in different combinations, in the vasculature, the musculature and the tissues of the handplate.

'Regeneration' of wing bud stumps of chick embryos and reactivation of Msx-1 and Shh expression in response to FGF-4 and ridge signals.

We examined systematically the ability of chick limb bud stumps to regenerate distal structures when fibroblast growth factor (FGF)-4 is applied. When amputations were made within 600 mu m of the tip and FGF-4 applied either posteriorly or both apically and posteriorly, outgrowth of stump tissues occurred and a virtually complete skeleton developed. 'Regeneration' of distal structures was correlated with reactivation of Msx-1 and Shh expression. At proximal amputation levels where FGF-4 did not lead to 'regeneration', neither Msx-1 nor Shh expression was induced. We also grafted cells from progressively more proximal levels of mouse limb buds to chick wing bud tips beneath the apical ridge and the pattern of reactivation of Msx-1 expression along the proximal distal axis of the limb buds was similar to that found in chick limb buds.

Gnot1, a member of a new homeobox gene subfamily, is expressed in a dynamic, region-specific domain along the proximodistal axis of the developing limb.

Limb development endures as an excellent model for pattern formation in vertebrates. We have identified Gnot1 as a member of a new homeobox gene subfamily. Gnot1 is expressed in a dynamic temporospatial distribution in the developing limb, initially correlating with regions destined to form distal structures and then becoming progressively more restricted to specific regions determined to give rise to wrist and ankle. Micro-surgical alteration of the developmental program of the limb reveals that Gnot1 is expressed in a position- and fate-dependent manner, responding both to signals from the apical ridge and the polarizing zone. Furthermore, Gnot1 activation by polarizing signals occurs temporally downstream of Hoxd gene activation, but well before the first appearance of condensations that will give rise to the carpus of the wrist. The features of Gnot1 expression suggest a role for this gene in regulating pattern formation during limb development.

FGF and genes encoding transcription factors in early limb specification.

SnR, twist and Fgf10 are expressed in presumptive limb territories of early chick embryos. When FGF-2/FGF-8 beads are implanted in chick flank, an ectopic limb develops and SnR is irreversibly activated as early as 1 h. Ectopic Fgf10 and twist expression are activated much later at 17 and 20 h, respectively. FGF-10 can also induce SnR, but much later, and in this case activation occurs simultaneously with that of twist and Fgf10 via the Fgf8- expressing ridge. Tbx-4 and Tbx-5 are expressed in leg and wing forming regions, respectively, in a similar pattern to SnR and twist. FGF-2 leads to ectopic expression of Tbx-4 and Tbx-5 as rapidly as ectopic expression of SnR, but the patterns of ectopic transcripts suggest that induction of SnR and Tbx gene expression occur via different pathways.

Expression of Drosophila trithorax-group homologues in chick embryos.

Mll, Brg1 and Brm are vertebrate homologues of Drosophila trithorax group (trxG) genes. We isolated chicken Mll cDNA clones, and examined patterns of Mll, Brg1 and Brm expression in chick embryos. All three genes were expressed from embryonic stage 2 onwards. Mll transcripts were just detectable in all tissues by in situ hybridization, with highest level in dorsal neural tube and notochord. Brg1 transcripts were readily detectable in all tissues, with highest levels in dorsal neural tube, dorsal trunk epithelium and limb bud epithelium and mesenchyme. Brm transcripts were more restricted, being found in dermomyotome, notochord, dorsal limb bud epithelium, eye and the roof and floor plates of the neural tube.

Limb development: an international model for vertebrate pattern formation.

Limb development is an excellent model for studying how patterns of differentiated cells and tissues are generated in vertebrate embryos. The cell interactions that mediate patterning have been discovered and, more recently, some of the molecules involved in these interactions have been identified. This has provided a direct link to genetics and thus to genes that cause human congenital limb defects.

Shh, Fgf4 and Hoxd gene expression in the mouse limb mutant hypodactyly.

The semidominant mouse mutation hypodactyly (Hd), caused by a deletion within the Hoxa13 gene, results in reduced digits; heterozygotes lack digit I in the hindlimb and homozygotes have only one digit on each limb. We investigated expression of Shh and Fgf4 signaling molecules involved in digit specification in mutant limb buds. Shh and Fgf4 are expressed in the posterior part of the limb buds as normal but expression may be slightly prolonged. The extent of digit reduction in hypodactyly is much more severe than in the Hoxa13 deficient mouse and resembles that in the Hoxa13(-/-)/Hoxd13(-/-) double mutant mouse. We found that the pattern of Hoxd13 and Hoxd11 transcripts was not markedly different in the mutant compared with the normal limbs even though the mutant limbs are narrower. Therefore Hoxd genes are transcribed as normal in the mutant. This makes it likely that the severe digit reductions in hypodactyly are caused by interference with Hoxd13 function at the protein level. Similar interactions between mutant and normal HOX gene products have been suggested to occur in the human semidominant disorder, synpolydactyly, caused by mutations in HOXD13.

Local origin of cells in FGF-4 - induced outgrowth of amputated chick wing bud stumps.

Urodele amphibians are the only vertebrates that can regenerate amputated limbs, even as adults. However, we have previously shown that amputated chick wing bud stumps can be induced to ((regenerate)) and to form a complete set of correctly-patterned skeletal elements, following implantation of beads soaked in fibroblast growth factor-4 (FGF-4). We have now performed Dil injection experiments to determine which cells contribute to FGF-4-induced chick wing bud ((regenerates)). We show that the FGF-4-induced outgrowth of the regenerating wing bud stump is comprised of mesenchyme cells that originate from a region within 200 microm of the FGF-4 bead, and that cells proximal to the bead move distally.

Syndactylies and polydactylies: embryological overview and suggested classification.

In 1978, Temtamy and McKusick classified isolated, non-syndromic polydactyly and syndactyly, using a logical anatomical approach, into five distinct types for each group. Since then, there have been considerable advances in the molecular embryology of the developing limb bud. These include the proposal that retinoic acid and/or related retinoids are the morphogens responsible for the morphogenetic gradient giving rise to anterior-posterior pattern formation of the limb bud, the suggestion that the HOX4 complex and other homeotic genes may also be involved in patterning, and a greater understanding of other mechanisms such as programmed cell death in the shaping of the final hand and foot. This paper briefly reviews the molecular embryology of limb development and outlines the 'end-organ responsiveness' of the limbs to a variety of single-gene mutations. An alternative classification of syndactylies and polydactylies is suggested. It is still too early to match specific defects to individual genes with precision, and it is obvious that many important developmental genes remain to be identified; nevertheless, it is envisaged that clues from molecular embryological studies will become increasingly more useful.

Alterations in Msx 1 and Msx 2 expression correlate with inhibition of outgrowth of chick facial primordia induced by retinoic acid.

Spatially-restricted expression domains of Msx 1 and Msx 2 in the developing chick face suggest that they may play a role in epithelial-mesenchymal interactions governing outgrowth of facial primordia. Retinoid application to developing chick faces reproducibly inhibits upper beak outgrowth but the lower beak is unaffected. In the normal face, high levels of Msx gene transcripts in upper and lower beak primordia correlate with regions of outgrowth. Following retinoid treatment, Msx 1 and Msx 2 transcripts are rapidly down-regulated in upper beak primordia where outgrowth is inhibited, but remain largely unchanged in lower beak primordia, where outgrowth is unaffected. Decreases in gene expression precede retinoid-induced morphological changes in the upper beak, suggesting that Msx gene products are involved in mediating the effect of retinoids on facial development.