Vertebrate Limb Bud Development-moving Towards Integrative Analysis of Org a No Genesis

The limb bud is an excellent model for gaining insights into the molecular mechanisms that control embryogen-esis. Over many decades, analyses of the limb bud have contributed substantially to our understanding of the common principles and cellular and molecular interac-tions that orchestrate organogenesis. Since the 1940s, the manipulation of chicken limb buds in so-called ‘cut and paste’ experiments has been used to define the groups of cells that control limb bud morphogenesis. The pheno-typic alterations in the limb skeleton provided an easy ‘read-out’ that is still used today. These classical studies led to the first models of limb bud development, which provided the conceptual framework for identifying important regulatory genes and interactions over the last two decades of the twentieth century. This identifica-tion was achieved by combining mouse genetics — using spontaneous and engineered loss- and gain-of-function mutants — with the experimental manipulation of chicken and mouse limb buds.

The initial genetic analysis focused mainly on elu-cidating the functions of a particular gene or pathway during limb bud development, but the challenge now is to understand how the genes and pathways interact to orchestrate limb organogenesis. Recent research has pro-vided insights into how cell specification and survival are coordinated with proliferation and determination during limb organogenesis. These studies have begun to show the highly dynamic nature of the regulatory networks that

control the orderly progression of limb organogenesis: outgrowth and patterning are controlled by a self- regulatory and robust signalling system that consists of interlinked feedback loops instead of independent mor-phogen signals. Therefore, this Review aims to take an integrative signalling systems point of view, rather than considering the two main limb bud axes separately.

First, we review the classical findings and models that provided the conceptual framework for understanding the development of the proximodistal (PD) and antero-posterior (AP) axes of the limb bud. Then, we discuss the signalling interactions that control the PD and AP axes and skeletal development. Based on recent data, we pro-pose an integrative model that considers the morpho-genesis of the two main limb axes together. We discuss this model with respect to the current understanding of the evolution of vertebrate paired appendages (fins and limbs) and congenital limb malformations in humans. As our review focuses on integrating AP with PD limb skeletal development, the interested reader is referred to other reviews for related topics, such as dorso-ventral limb axis specification and apical ectodermal ridge (AER) formation1, transcriptional regulation of limb bud identity and development2,3, and paired appendage evolution4.

Although the current knowledge of the regulatory networks contains too many gaps to draft a comprehen-sive systems model, we seek to provide the context for

Developmental Genetics, Department of Biomedicine, University of Basel, Mattenstrasse 28, CH‑4058 Basel, Switzerland.Correspondence to R.Z. e‑mail: [email protected]:10.1038/nrg2681

SpecificationThe process preceding determination during which a cell acquires its fate. The exposure of specified cells to different signals might alter their fates; the fate of specified cells is not fixed (unlike determined cells).

DeterminationWhen cell fate is fixed so that the cell will initiate differentiation into the specified cell type even if the cell is isolated or transplanted into a different environment or tissue. Determination occurs before the appearance of cell-type-specific morphological characteristics, but is often closely followed by the initiation of differentiation.

Vertebrate limb bud development: moving towards integrative analysis of organogenesisRolf Zeller, Javier López‑Ríos and Aimée Zuniga

Abstract | The limb bud is of paradigmatic value to understanding vertebrate organogenesis. Recent genetic analysis in mice has revealed the existence of a largely self-regulatory limb bud signalling system that involves many of the pathways that are known to regulate morphogenesis. These findings contrast with the prevailing view that the main limb bud axes develop largely independently of one another. In this Review, we discuss models of limb development and attempt to integrate the current knowledge of the signalling interactions that govern limb skeletal development into a systems model. The resulting integrative model provides insights into how the specification and proliferative expansion of the anteroposterior and proximodistal limb bud axes are coordinately controlled in time and space.

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PatterningThe process in which the positions and identities of cells with different fates are laid down.

Apical ectodermal ridgeA specialized epithelium that is required for limb bud outgrowth. It runs along the distal tip of the limb bud and expresses several fibroblast growth factors. During the initiation of limb bud development, the ridge forms at the dorsoventral interface of the limb bud ectoderm.

Epithelial–mesenchymal feedback loopSignalling interactions between an epithelium and the adjacent mesenchyme. These interactions of different signals from both compartments form a closed feedback loop. The development of many organs is controlled by epithelial–mesenchymal feedback loops.

Fate mappingFollowing the fates and progeny of cells during embryonic development by marking them with lipophilic dyes or recombinant retroviruses, or by using molecular genetic tools that result in permanent expression of an inert marker gene, such as β-galactosidase or GFP.

FateThe fate of a cell is normally dependent on specific inductive signals. After their fate is determined, cells will normally follow a specific developmental sequence towards differentiation into a particular cell type.

a holistic rather than deconstructive approach to limb organogenesis. in particular, there is an urgent need to combine molecular genetics and experimental manipu-lation with quantitative biology and modelling — that is, to apply systems biology approaches to the study of limb and embryonic development.

Proximodistal limb bud developmentlimb buds derive from the flank mesenchyme, and their outgrowth is initiated at defined positions along the embryonic axis (FIG. 1a). limb buds consist of an ectodermal pocket that envelops the proliferating mes-enchyme. The AER is a specialized epithelium that runs along the distal limb bud tip. The PD limb bud axis is defined by the primary direction of outgrowth and results in the formation of the stylopod, zeugo-pod and autopod (FIG. 1b). The AP axis is defined by the sequence of digits 1 to 5 (thumb to little finger). in this section, we describe the classical experiments that led to the proposition of the ‘progress zone’ model of PD limb bud development. Subsequent analyses resulted in refined mechanistic models that describe the molecular and cellular interactions that control PD limb axis devel-opment and that are also consistent with findings from non-molecular studies.

Classical studies and models. PD limb bud outgrowth is remarkably rapid: mouse limb buds expand by ~1,300 μm between embryonic days 9.5 and 12. The three limb skel-etal domains (FIG. 1b) are determined during this time. Removal of the AER from the wing buds of chicken embryos showed that the AER produces the signals that promote PD outgrowth. These experiments led to the formulation of the progress-zone model5,6 (FIG. 1c). This model postulates that signals from the AER keep cells in the distal-most limb bud mesenchyme — the progress zone — in a proliferative and undetermined state. The identities of cells along the PD axis are specified by a ‘clock-type’ mechanism as they exit the progress zone — that is, the cell identities depend on the time of exit.

Molecular and mechanistic insights. After the formula-tion of the conceptual model, the next issue was to iden-tify the molecular signals produced by the AER. members of the fibroblast growth factor (Fgf) gene family encode the crucial signals7. Fgf8 is expressed from the time of specification of the AER onwards. Fgf4, Fgf9 and Fgf17 are activated subsequently in the posterior AER, and their expression expands in an anterior direction during the progression of limb organogenesis8,9. The importance of AER-derived FGF signals (AER-FGFs) was shown by the ability of beads impregnated with FGFs to restore limb bud outgrowth and patterning in chicken limb buds from which the AER had been removed7,10.

By contrast, the origin and nature of the signal(s) that initiates limb bud formation remains unknown. However, implantation of FGF-soaked beads into the flank mesenchyme of early chicken embryos induces the formation of ectopic limbs11,12. WnT ligands, which are normally expressed in the presumptive limb terri-tories in chicken embryos, also induce ectopic limbs12;

this study implicated WnT2b and WnT8c in the activa-tion of Fgf10 expression and AER formation. However, these WnT ligands are not expressed during limb bud induction in mouse embryos13. During these early stages, Fgf10 and bone morphogenetic protein 4 (Bmp4) are expressed by the limb bud mesenchyme. inactivation of these genes in mouse embryos disrupts the formation of a fully functional AER and the survival of the mes-enchyme but does not disrupt limb bud induction14,15. in response to mesenchymal FGF10 signalling, Fgf8 is activated in AER progenitors, which results in the estab-lishment of a growth-promoting epithelial–mesenchymal feedback loop (e–m feedback loop) between AER-derived FGF8 and mesenchymal FGF10 (ReF. 16).

Genetic analysis of the Fgf genes that are expressed in the AER showed that Fgf8 alone is essential for limb bud development. limb buds that lack Fgf8 expression in the AER are smaller than normal, and activation of sonic hedgehog (Shh) expression (see below) is delayed. This delay results in the loss of certain skeletal elements8. mouse limb buds that lack expression of Fgf4, Fgf9 and Fgf17 in the AER develop normally9. By contrast, limb bud development is completely disrupted when Fgf4 and Fgf8 are inactivated together17. Furthermore, transient expression of Fgf4 and Fgf8 in early limb buds is suf-ficient to specify the entire PD axis, but the subsequent proliferative expansion is disrupted17.

These genetic analyses established that AER-FGFs specify the PD axis at an early stage and that the speci-fied progenitor pools are progressively expanded9. Furthermore, fate mapping experiments in chicken limb buds indicated that PD identities are specified early and the specified progenitor pools expand sequentially18. Therefore, genetic ablation or removal of the AER at an early stage would eliminate most of the specified mes-enchymal progenitors, whereas later removal of the AER would eliminate the more distal progenitors that had not yet expanded.

These conclusions are difficult to reconcile with the progress-zone model. They do, however, fit with an alternative model — the two-signal model — which is discussed below.

The two-signal and differentiation-front models. Analysis of chicken limb bud development has shown that retinoic acid (RA) induces proximal cell identity in the limb bud mesenchyme and that AER-FGFs, which antagonize RA signalling, induce distal cell identity19. The authors of this study concluded that RA induces Meis1 and Meis2 expression in the proximal stylopod territory and that AER-FGFs lead to the activation of homeobox A11 and A13 (Hoxa11 and Hoxa13), which is indicative of the specification of more distal fates that will give rise to the zeugopod and autopod as limb bud outgrowth progresses19,20 (FIG. 1d). indeed, inactivation of retinaldehyde dehydrogenase 2 (Raldh2), which is required for RA synthesis in mouse embryos, disrupts limb bud initiation; this phenotype is rescued by sup-plying exogenous RA21. A further study indicated that a graded distribution of RA in mouse limb buds could be enhanced by active RA degradation in the distal

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Figure 1 | Proximodistal limb bud axis development. a | Scanning electron microscopy image of a mouse embryo at gestational day 10.5. The forelimb bud forms at the level of the heart; hindlimb development (not shown) is delayed by about 12 hours and occurs at the level of the kidneys. The enlarged inset shows the forelimb bud with the two main limb bud axes indicated. The apical ectodermal ridge (AER) is indicated in green. b | The skeletal elements of a human arm. The stylopod gives rise to the most proximal limb skeletal element, the humerus. The zeugopod forms the radius (anterior) and ulna (posterior). The distal autopod forms the wrist bones (carpals), palm bones (metacarpals) and digit bones (phalanges). The scapula and clavicle do not derive from the limb bud. c | The progress zone (PZ) model was formulated to explain the limb skeletal phenotypes that result from the manipulation of chicken limb buds5. It was proposed that the mesenchyme that underlies the AER contains unspecified progenitors (the PZ is indicated by black stripes)6, the fates of which are controlled by AER signals. As limb bud outgrowth progresses distally, proximal cells no longer receive AER signals. The time of their ‘exit’ from the PZ determines their proximodistal (PD) identity (that is, there is ‘clock-type’ specification). Mesenchymal cells that exit early generate proximal elements, whereas cells that remain in the PZ for longer form more distal structures. d | Based on molecular analysis of chicken limb bud development, the two-signal model was proposed19. During the onset of limb bud development, the proximal region (blue) is probably specified by retinoic acid (RA) signalling from the flank, and the distal region (orange) is specified by AER-derived fibroblast growth factor (AER-FGF) signalling. The zeugopod arises from the more proximal distal cells, and the autopod primordia is formed by the most distal mesenchymal cells9. e | The differentiation-front model25 postulates that PD identities are determined as the proliferating mesenchyme leaves the undifferentiated zone — that is, when the mesenchyme is no longer under the influence of AER-FGF signalling. After cells have crossed the differentiation front (blue wavy line) they only express genes that mark the identity of a particular segment (for example, Meis1 expression in the stylopod territory, homeobox A11 (Hoxa11) expression in the zeugopod territory and Hoxa13 expression in the autopod territory). Part a: image courtesy of O. Michos and Central Microscopy Facility, University of Basel, Switzerland.

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DifferentiationWhen cells activate genes that result in the appearance of cell-type-specific characters. During embryonic development, groups of cells that have been determined will initiate differentiation into specific functional tissues.

OrganizerA small group of embryonic cells that have the ability to influence the fate, survival and/or proliferative potential of other cells. Grafting organizer cells to ectopic positions will induce ectopic structures (for example, mirror-image duplications of digits). Organizer cells secrete signals or antagonists that can act as morphogens.

mesenchyme22. Therefore, it was proposed that opposing activities of RA and AER-FGFs in the proximal and dis-tal zones, respectively, specify the PD limb bud axis at early developmental stages19–21 (FIG. 1d). This is known as the two-signal model.

The two-signal model is supported by genetic analysis of AER-FGF functions9 (see above). However, this and other studies revealed that the expression domains of the Meis genes, Hoxa11 and Hoxa13 do not accurately define the prospective zeugopod and autopod territories, prob-ably owing to the dynamic regulation of their expression domains in early development9,23. Furthermore, not all studies agree that RA signalling from the flank mesen-chyme is required for specification of the PD limb bud axis. For example, a recent analysis of mouse embryos that lack Raldh2 and/or Raldh3 and were rescued by exogenous RA showed that RA signalling enables limb bud formation by inhibiting Fgf8 expression in the intermediate mesenchyme24. Therefore, it is still a mat-ter of debate whether RA is indeed an essential proximal signal, as proposed by the two-signal model19–21.

in an attempt to reconcile the molecular data with the classical AER-removal experiments, the differentiation front was proposed as a dynamic description of the molecular and cellular events that occur during the pro-gression of PD axis development25 (FIG. 1e). The descrip-tion of the differentiation front incorporates the main features of the two-signal model. it postulates that the early limb bud mesenchyme is fated to give rise to proxi-mal structures, but that this ‘default’ is modified by AER-FGF signals. These signals induce the specification of distal fates, as indicated by Hoxa11 and Hoxa13 expres-sion. AER-FGFs keep the distal mesenchymal cells in a proliferative, undifferentiated state, whereas the proxi-mal cells are progressively determined, as indicated by the expression of segment-specific marker genes and the initiation of chondrogenic differentiation. The border between the determined proximal cells and the specified distal cells defines the differentiation front, the position of which becomes more distal during the progression of limb bud development.

A recent study has shown that ectodermal WnT (e-WnT) signals interact with AER-FGFs; together these signals keep the distal-most mesenchyme (~250 μm from the tip) in an undifferentiated, proliferative state26. The mesenchymal cells outside the range of either of these signals proliferate less, initiate SRY-box containing 9 (Sox9) expression and form the cartilage models that prefigure the limb skeleton. loss or gain of canonical WnT signal transduction in mouse limb buds results in ectopic or reduced expression of Sox9, respectively27. in limb buds, Sox9 marks the chondrocyte lineage and functions in chondrocyte differentiation. Also, a recent repeat of the classical X-ray irradiation studies (previ-ously interpreted in favour of the progress-zone model) showed that the molecular markers of PD axis devel-opment are not significantly affected by irradiation23. Rather, the chondrogenic precursors that are differen-tiating at the time of X-ray radiation are eliminated by apoptosis, which results in loss of the corresponding skeletal elements.

Anteroposterior limb axis specificationin this section, we first discuss the classical tissue grafting experiments in chicken limb buds that resulted in the identification of the signalling centre that controls AP axis development. We then describe how this led to the identification of the relevant morphogen signal and how recent studies have revealed the complex interac-tions that control specification and expansion of the AP limb mesenchymal progenitors.

From the French flag to sonic hedgehog. The transplanta-tion of small pieces of mesenchyme to ectopic locations in chicken wing buds led to the identification of an organizer located in the posterior mesenchyme. This limb bud organizer is called the polarizing region or zone of polar-izing activity (ZPA) (FIG. 2a) and is defined by its poten-tial to instruct limb bud mesenchymal cells with respect to their AP fates. For example, grafts of ZPA cells (≥150 cells) to an anterior position in limb buds induces com-plete mirror image digit duplications5,28. in an attempt to provide a mechanistic understanding of AP limb bud development, Wolpert formulated the French-flag model29, which postulated that ZPA cells secrete a mor-phogen that forms a gradient across the limb bud (BOX 1) and specifies the identities of mesenchymal cells by three specific thresholds (corresponding to the three wing dig-its and likened to the colours of the French flag (FIG. 2a). A decade-long hunt for Wolpert’s morphogen resulted in the identification of sonic hedgehog (SHH) as the signal produced by the ZPA30. Anterior grafts of Shh-expressing fibroblasts produce mirror-image digit duplications, and genetic inactivation of Shh in mouse embryos causes the loss of posterior identities (the ulna and digits 2 to 5)31,32. The most anterior digit — the thumb, digit 1 — does not require SHH signalling. its development depends on the Sal-like 4 (SAll4), T-box 5 (TBX5) and HOX transcriptional regulators33,34.

Zone of polarizing activity establishment and sonic hedgehog signalling. in mice, a number of transcrip-tional regulators have been implicated in the control of Shh expression at the posterior limb bud margin. in par-ticular, loss-of-function mutations in heart and neural crest derivatives 2 (Hand2) and 5′-located Hoxd genes (5′-Hoxd) disrupt Shh activation in limb buds (ReF. 35; A. Galli and R.Z., unpublished observations). in agree-ment, biochemical analysis showed direct interaction of HAnD2 and 5′-HOXD with the upstream cis-regulatory sequences that control Shh activation in limb buds (ReF. 36; A. Galli and R.Z., unpublished observations). The posterior restriction of Hand2 and 5′-Hoxd expres-sion in early limb buds requires Gli3 activity (FIG. 2b). Gli3 pre-patterns the nascent limb bud and restricts Shh activation37. The position of the Shh expression domain at the posterior limb bud margin is controlled by many factors, including Tbx genes, RA signalling by the flank (which might regulate Hand2 and 5′-Hoxd expression) and Fgf8 expression in the AER8,21,38 (FIG. 2b).

manipulation of SHH signalling in chicken and mouse limb buds provided good evidence for its graded signalling activity, as postulated by the French-flag

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Figure 2 | Anteroposterior limb bud axis development. a | The French-flag model29 can explain the results of manipulating the chicken limb bud organizer (zone of polarizing activity (ZPA), which is located in the posterior limb bud mesenchyme). This model proposes that the ZPA secretes a morphogen that diffuses across the limb bud to generate a spatial gradient. The identities of the three digits (likened to the colours of the French flag) are specified by threshold levels of the morphogen. b | The gene network that restricts the activation and maintenance of sonic hedgehog (Shh) expression to the posterior limb bud mesenchyme. c | Specification of anteroposterior identities by a spatial and temporal gradient of SHH signalling46,47. As cells cease to express Shh, they exit the ZPA. The expanding population of cells derived from Shh-expressing cells (Shh descendants) displaces non-ZPA cells (which are specified by long-range SHH signalling) towards the anterior. Shh descendants give rise to the ulna, digit 4 and digit 5, and contribute to digit 3. Cells that give rise to digit 2 and parts of digit 3 are specified by long-range SHH signalling. The humerus, radius and digit 1 are specified in an SHH-independent manner. 5′-HOXD, 5′-located homeobox D; FGF8, fibroblast growth factor 8; GLI3R, repressor form of GLI3; HAND2, heart and neural crest derivatives 2; RA, retinoic acid; TBX, T-box.

PolydactylyFrom the Greek for ‘many fingers’. Limb skeletal phenotypes that result in the formation of additional digits. There are three types of polydactylies: postaxial (affecting the little finger), preaxial (affecting the thumb) and central (affecting the ring, middle and index fingers).

CylopamineA small molecule that inhibits all hedgehog signal transduction by binding to the smoothened seven-transmembrane receptor.

model. The range of SHH activity was estimated as being approximately half of the early limb bud (or ~300 μm from implanted beads). SHH induces or upregulates the expression of target genes, such as the Gli1 transcriptional activator and the inhibi-tory patched 1 (Ptch1) receptor39. Furthermore, the constitutive processing of Gli3 to its repressor form (Gli3R) is inhibited in response to SHH signalling; the full-length activator form (Gli3A) functions as a positive transcriptional regulator 40. The anterior limb bud contains high Gli3R and low Gli3A levels, and the converse is true close to the SHH signalling domain in the posterior region. Genetic inactiva-tion of Gli3 in mice and humans results in preaxial polydactylies41,42. As the same polydactylies are observed in mouse limb buds that lack both Shh and Gli3, one function of SHH must be to counteract Gli3R-mediated repression to enable the progression of limb organogenesis43,44. SHH is also modified by cholesterol, and this modification restricts the range of SHH sig-nalling. SHH lacking this modification is more dif-fusible, which results in anterior digit duplications45. These preaxial polydactylies indicate that digit 2 — which is the SHH-dependent digit furthest from the SHH source — is most sensitive to increased diffu-sion. in agreement, genetic fate-mapping studies have shown that only digit 2 depends entirely on long-range SHH signalling46,47.

When and how are digit identities determined? Although Wolpert’s morphogen gradient is depicted in a static manner (FIG. 2a), numerous studies have shown that the requirements, interactions and functions of gene products can vary over time. With respect to autopod patterning, experimental evidence indicates that the cells that contribute to digit 2 and parts of digit 3 are specified by long-range SHH signalling, and the posterior digits 3 to 5 derive from mesenchymal cells that have previously expressed Shh as part of the ZPA46,47 (FIG. 2c). cells that derive from Shh-expressing cells (Shh descendants) soon after Shh activation contribute to digit 3, and descend-ants of cells that have expressed Shh for longer contrib-ute to progressively more posterior digits. The resulting gradient of autocrine exposure to SHH (FIG. 2c) is likely to provide cells with a temporal memory that contrib-utes to the determination of their identities47. in agree-ment with this conclusion, brief exposure to SHH is sufficient to specify anterior but not posterior digits48.

Sonic hedgehog promotes proliferation of the limb bud mesenchyme. The studies discussed above indicated that SHH-mediated specification of digit identities is linked to proliferation. indeed, cylopamine-mediated inhibition of SHH signal transduction in chicken wing buds dis-rupts the cell cycle and proliferative expansion, resulting in loss of posterior digit identities49. By contrast, the spe-cific inhibition of cell proliferation causes an immediate

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Box 1 | Morphogens: more than gradients

Morphogens are defined as diffusible molecules that are secreted by cells with so-called ‘organizer’ properties, as originally defined by transplanting the Mangold–Spemann organizer in gastrulating amphibian embryos85. Diffusion of morphogens results in a morphogen gradient. Gradients can be regulated by a source, which controls morphogen levels, and a sink, which controls morphogen degradation. Cells respond to particular levels of morphogens, as postulated in the French-flag model29 (FIG. 2a).

A paradigmatic model of morphogen activity in vertebrate embryos is sonic hedgehog (SHH)-mediated dorsoventral patterning of the developing neural tube86. SHH is secreted by cells of the ventral neural tube floorplate and controls the dorsoventral identity of neural progenitors as it spreads through the lumen of the neural tube. SHH signal transduction through the primary cilia of progenitor cells induces both positive and negative transcriptional regulation (for details, see ReF. 86). The response of neural tube cells to SHH is desensitized over time owing to upregulation of the inhibitory receptor patched 1 (PTCH1). Therefore, there is an adaptation mechanism by which cells integrate the duration and concentration of SHH signalling87.

These and other studies have revealed that the spread of morphogens and the response to morphogens are controlled at multiple levels. The signal itself is controlled by processing and/or modifications that affect its intracellular trafficking, secretion and diffusion and its interactions with the extracellular matrix, receptors and regulators86,88. The shape of the gradient and the cellular responses are modulated by the activation of cell surface proteins that enhance or inhibit signal reception and/or secreted secondary signals that promote lateral inhibition86,89.

The kinetic parameters of the morphogen gradients are currently best understood for wing imaginal disc development in Drosophila melanogaster larvae. Genetic analysis has established that the Decapentaplegic (DPP; an orthologue of vertebrate bone morphogenetic proteins (BMPs)) signal acts as a long-range morphogen in the wing imaginal disc epithelium and that DPP controls disc growth and patterning90. Molecular analysis in combination with mathematical modelling has revealed that DPP has a half-life of ~45 minutes and that it diffuses at a rate of ~0.1 μm2/s. This results in a gradient that spans 30–40 cells91. By contrast, the Wingless and Hedgehog morphogens (orthologues of vertebrate WNTs and SHH) form short-range gradients spanning 10–15 cells91,92. One general lesson from these studies is the need to base mathematical models on experimentally determined parameters that are obtained from analysis of the endogenous signalling networks. Furthermore, the fact that signalling gradients and their reception are dynamic in time and space — owing to proliferative enlargement of the target cell population, cellular apoptosis, cell polarization and/or modulation of cellular responsiveness — has to be incorporated into mathematical models of morphogen gradients and signalling interactions.

but transient loss of SHH signalling, which results in the formation of only posterior digits. This is because all of the reduced autopod primordium is exposed to high SHH levels49. Temporally controlled genetic inactivation of Shh in mouse limb buds indicates that SHH-mediated specification of AP digit identities happens during early limb bud development, and that subsequently SHH is required for proliferation of the specified progenitors50. in other words, the two functions of SHH — that is, the specification of AP digit identities and the control of cell numbers — seem to be temporally uncoupled. inactivation of Shh at progressively earlier time points causes loss of digits in the sequence digit 3, digit 5, digit 2, digit 4, which is difficult to reconcile with a simple mor-phogen gradient. Rather, it seems to reflect the temporal sequence in which the chondrogenic condensations that demarcate particular digits appear (digit 4 before digit 2, then digit 5 and lastly digit 3)50. A previous analysis had concluded that the posterior digit 5 differentiates last51, in agreement with the proposal that it is formed

from the cells that have expressed SHH for the longest period of time47. Finally, the apparent uncoupling of specification and growth in mice differs from the pro-posed morphogen-controlled growth mechanism that regulates chicken digit morphogenesis. This discrep-ancy could be a consequence of the fact that chicken wing buds form only 3 digits and only digit 4 is derived from Shh-expressing cells49 (see ReF. 52 for a more extensive discussion).

Interlinked signalling feedback loopsAlthough most of the studies discussed so far have focused on analysing the morphogenesis of one particu-lar limb bud axis, it has long been noted that the AER is required to maintain the ZPA, and vice versa. This indicates that the two signalling centres, and thereby the patterning of the axes, are linked28. in this section, we discuss the discovery of the e–m feedback signalling loop that interlinks the two signalling centres. This e–m feedback loop is part of a more complex signalling sys-tem that coordinately controls AP and PD limb bud axis development in a largely self-regulatory manner.

The molecular basis for the interplay between the AER and ZPA was discovered by the observation that beads soaked in FGFs maintain both Shh expression and outgrowth after AER removal53,54. in addition, the BmP antagonist gremlin 1 (GREm1) is required in the subectodermal mesenchyme to relay the SHH sig-nal to the AER and to promote the upregulation of Fgf expression in the AER55,56. Together, these signals define the SHH–GREm1–FGF e–m feedback loop. inactivation of Grem1 in mouse embryos disrupts the expansion of the distal limb bud and the specification of AP identi-ties, which indicates that GREm1-mediated reduction of BmP activity is essential for the progression of limb bud development55.

However, additional genetic studies revealed that mesenchymal BmP4 signalling is required to induce a functional AER and initiate Grem1 expression in the posterior mesenchyme14,57 (FIG. 3a). increased GREm1 antagonism decreases mesenchymal BmP activity, which enables SHH–GREm1–FGF e–m feedback sig-nalling and distal progression of limb organogenesis in a largely self-regulatory manner14 (FIG. 3b). The SHH–GREm1–FGF e–m feedback loop is self-terminated as the expanding population of Shh descendants is refrac-tory to Grem1 expression: the expansion generates an increasing gap that moves the Grem1 expression domain ‘out of reach’ of SHH signalling58 (FIG. 3c). Genetic analy-sis in the mouse limb bud mesenchyme showed that increased AER-FGF signalling triggers an inhibitory loop that contributes to the shutdown of Grem1 expres-sion and e–m feedback signalling59. mathematical mod-elling of these interactions has identified the successive and differential transcriptional regulation of Grem1 by BmP4, SHH and AER-FGFs as the crucial node in this self-regulatory signalling system14. interestingly, mes-enchymal BmP activity changes with the kinetics of a transitory feedforward loop: it is initially high and is required for AER formation, then it lowers and regu-lates the AER length during proliferative expansion,

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Figure 3 | interlinked feedback loops define a self-regulatory limb signalling system. The mesenchymal bone morphogenetic protein (BMP4; light blue), sonic hedgehog (SHH; red), gremlin 1 (GREM1; purple) and apical ectodermal ridge-derived fibroblast growth factor (AER-FGF; green) expression domains during mouse limb organogenesis are indicated schematically. The interlinked signalling feedback loops14 that operate at each stage are shown as solid lines. Broken lines indicate inactive loops. a | Initiation phase: BMP4 upregulates Grem1 expression in a fast initiator loop (~2 h loop time). Shh expression and signalling are activated independently of GREM1 and AER-FGFs. b | Propagation phase: the establishment of loops that control the distal progression of limb bud development. SHH predominantly upregulates Grem1 expression. GREM1 reinforces AER-FGF and zone of polarizing activity-derived SHH (ZPA-SHH) signalling by an epithelial–mesenchymal feedback loop (with a loop time of ~12 h). The activity of the fast BMP4–Grem1 initiator module is low. However, this low BMP activity controls the length of the AER (not shown). c | Termination phase: the widening gap between ZPA-SHH signalling and the Grem1 expression domain, together with the onset of AER-FGF-mediated inhibition of Grem1, terminates the signalling system. As a consequence, BMP4 activity is likely to increase again.

and finally it rises again and is likely to participate in the determination of digit identities as the system is terminated (see below).

An integrative model for limb organogenesisThe studies discussed above reveal the complexity of the interactions that link specification of the PD and AP limb bud axes with outgrowth, determination and chondrogenic differentiation. To make progress in the identification and understanding of the systems of morphological regulation, it is important to con-sider AP and PD limb bud axis development together, rather than as separate developmental processes, as is currently the norm. Here, we set out an integrative model that is based on the literature discussed and that focuses on the mechanisms and interactions that are most likely to be important. in addition to both limb bud axes, temporal and spatial aspects of gene function must be considered; a gene product might be essential at one stage, but later might be dispensable or have a different function14 (for example, see BmP4 in FIG. 3). in developing our integrative four-step model, we have realized that there are still substantial gaps in our molecular and cellular understanding of how limb bud development is orchestrated. in particular, certain key findings are still disputed (for example, the role of RA in limb bud initiation, discussed above) or have only

been evidenced in one particular species (for example, the roles of BmPs in the determination of digit iden-tities, see below). Therefore, this integrative model is imperfect, but it should provide an interesting and useful framework for developing a holistic approach to study the regulatory systems and interactions that control limb organogenesis.

Initiation and early specification. During limb bud initiation (FIG. 4a), the two main signalling centres are established. it is possible that the nascent limb bud mes-enchyme is initially of proximal and/or anterior char-acter19,60. The most distal and posterior cell identities are likely to be specified first by the onset of FGF8 sig-nalling in the AER (FIG. 1d) and Gli3-mediated restric-tion of Hand2 and 5′-Hoxd gene expression. These Gli3–HAnD2 mediated interactions pre-pattern the limb bud along its AP axis and activate SHH signal-ling at the posterior limb bud margin37. Alternatively, AP polarity might be transferred from the embryonic body axis (head to tail) to the limb field, as is the case for the dorsoventral limb bud axis61,62. To understand these initiating events, it will be essential to identify the mechanisms that activate and restrict the expression of key regulatory genes in the lateral plate and limb field mesenchyme (for example, Gli3, Hand2, 5′-Hoxd, Bmp4 and Fgf10).

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AER-FGFgradient

pre-AER(FGF8+WNT)

Humerus

d4

d2

d1

d5

d3

Ectoderm(e-WNT)

Prox

imal

Prox

imal

1

2

5

3

Radius

Ulna 4 U

R

~100 µm

Ectoderm(e-WNT)

1

2

5

3

4

DistalRARA

Distal

~250 µm

>100 µm

Anterior

Posterior

c Proliferative expansion and differentiation front d Late determination of digit identities

Ulna

Radius

Zeugopod Autopod

Prospective ZPA

e-WNT range

Proximal

Gli3

5′-Hoxd + Hand2

~300 µm

Zeugopodcross-section

Autopodcross-section

3

e-WNT range

ZPA descendants

AER range (FGF+WNT)

Sox9

ZPA-Shh

Zeugopod

Autopod

Stylopod

Long-rangeSHH signalling

2

4

Shh

PFRPositional informationfrom ID to PFR

Zeugopodcross-section

Autopodcross-section

b Early specification a Initiation

~250 µmAnterior Anterior

ZPA-SHH gradientPosterior Posterior

Figure 4 | An integrative model for limb bud development. a | During limb bud initiation, apical ectodermal ridge-derived fibroblast growth factor (AER-FGF) signalling is upregulated and zone of proliferating activity-derived sonic hedgehog (ZPA-SHH) signalling is initiated. The nascent mesenchyme is likely to be of proximal character and entirely exposed to ectodermal WNT (e-WNT) signalling (right panel). Retinoic acid (RA) signalling derived from the flank might define the proximal region and AER-FGF signalling defines the distal region. Restriction of heart and neural crest derivatives 2 (Hand2) and 5′-located homeobox D (5′-Hoxd) expression establishes posterior identity. In mouse forelimb buds this occurs from about embryonic day 9 (E9) onwards. The thick black arrows in the left panel indicate the approximate position of the transverse section shown in the right panel. b | Early specification: AER-FGF and ZPA-SHH signalling gradients act over a distance of ≥250 μm to specify distal and posterior identities (probably from E9.5 onwards in mice). Three domains of SHH signalling along the anteroposterior (AP) axis are likely to be specified at this stage: the anterior SHH-independent domain, which gives rise to the radius and digit 1 (d1); the SHH-dependent anterior domain, which gives rise to d2 and d3; and the posterior domain, which gives rise to the ulna and digits d3 to d5. c | Proliferative expansion of the limb bud occurs under the influence of AER-FGF, e-WNT and ZPA-SHH signalling (probably from E9.75 onwards in mice). As outgrowth progresses, the distal mesenchymal cells remain in an undifferentiated state, whereas core mesenchymal cells (no longer in range of these signals) activate SRY-box containing 9 (Sox9) expression and initiate chondrogenic differentiation (see also FIG. 1e). Concurrently with distal progression of the differentiation front, AP limb skeletal identities are determined in proximal to distal sequence (radius and ulna followed by the digits). The left panel shows a mouse forelimb bud at E11.5 with Sox9-positive condensations of humerus, ulna, radius (proximal to the differentiation front) and digits d2 and d4. The zeugopod cross-section shows the ongoing differentiation of the ulna and radius proximal to the differentiation front. The autopod cross-section shows the distal-most mesenchyme, which remains in an undifferentiated state under the influence of AER signalling (green dots; distal to the differentiation front). d | The proposed late determination of digit identities (from E12 onwards) is likely to occur as a consequence of integrating the ‘memory’ that autopod progenitors have acquired during proliferation (FIG. 2c) with BMP signalling from the interdigital mesenchyme (ID), which acts on the phalanx-forming regions (PFRs).

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Activation of AER-FGF signalling and ZPA-derived SHH (ZPA-SHH) signalling might induce the specifi-cation of the two main limb bud axes by morphogen gradients that act across at least half of the early limb bud. This proposal is well supported by genetic analyses in mice, which indicate that during early stages distal versus proximal and posterior versus anterior identi-ties are specified by AER-FGF and ZPA-SHH signalling, respectively (specification phase)9,50 (FIG. 4b). Although axis polarity is specified, cell identities are not deter-mined at this stage, as shown by lineage analysis that revealed extensive cell mingling and a lack of lineage restriction in early mouse limb buds63. How cell fates are specified is poorly understood, but the sequen-tial activation of transcriptional regulators belonging to the Hoxa, Hoxd and Tbx gene families demarcate defined limb bud regions2,3. At this early stage, BmP activity decreases and the SHH–GREm1–FGF e–m feedback loop is initiated and promotes proliferative expansion (FIG. 3b).

Proliferative expansion, determination and differentia-tion. The expansion, determination and differentiation phase seems to be the longest and most dynamic period of vertebrate limb bud development. coordinated pro-liferation along both axes (FIG. 4c) expands the progeni-tor pools, which give rise to the stylopod, zeugopod and autopod primordia. This expansion is primarily controlled by a self-regulatory system that involves the ZPA-SHH, AER-FGF and e-WnT growth promoting signals. Fate mapping analysis has indicated that prolif-eration rates are largely homogenous along the AP axis in both chicken and mouse limb buds63,64. low but per-sistent mesenchymal BmP activity (not shown in FIG. 4c) restricts the length of the AER, as shown by the observa-tion that inactivation of BmP during this developmen-tal period in mouse embryos causes an expansion of AER-FGF signalling and digit polydactylies14,65.

As limb bud outgrowth proceeds distally, the proxi-mal core mesenchyme is no longer under the influence of AER-FGF and e-WnT signalling. This leads to the initiation of Sox9 expression and chondrogenic differ-entiation25 (FIG. 4c). Proximal to the differentiation front, PD and AP identities are determined and differentiation is initiated (FIG. 4c, zeugopod cross-section). By contrast, distal mesenchymal cells remain undifferentiated owing to their continued exposure to AER signalling (FIG. 4c, autopod cross-section). As proliferative expansion and differentiation progress distally, the expansion of the anterior autopod primordium gives rise to digit 1 (which is SHH-independent) and digit 2 and the anterior part of digit 3 (which are SHH-dependent). The progres-sive expansion of the Shh descendants will contribute to digit 3 and generate digits 4 and 5 (ReFS 47,50). The massive proliferative expansion ends by self-termination of the limb bud signalling system (FIG. 3c).

Evidence for late determination of digit identities. currently it is not clear when digit identities are deter-mined or fixed. Experimental manipulation of chicken limb buds has provided evidence in favour of a late,

BmP-dependent mechanism. These experiments indi-cated that BmPs act downstream of SHH in digit pat-terning66, but mouse genetic analysis in combination with modelling has indicated that BmP activity is low during the predominantly SHH-dependent phase of autopod development14 (FIG. 3b). BmP activity increases during the termination of SHH–GREm1–FGF feedback signalling and, indeed, the manipulation of chicken leg buds indicates that digit identities are likely to be determined in a BmP-dependent manner during these late stages67 (FIG. 4d).

BmP signalling from the interdigital mesenchyme has been proposed to act on the distal tips of the con-densing digit primordia — the so-called phalanx-forming regions (PFRs) — which are located directly under the AER67 (FIG. 4d). The PFRs are characterized by unique BmP signal transduction signatures, which are higher in posterior PFRs than in anterior PFRs, with the exception of digit 4 (ReF. 68). The length and number of phalanges in a particular digit primordium is regulated by AER-FGF signalling in chicken limb buds69. However, as the memory that progenitors have accumulated during proliferation needs to be consid-ered47, BmP-dependent digit determination might serve as a ‘proofing’ mechanism rather than the mechanism that would determine digit identities per se. it is impor-tant to note that there is currently no genetic evidence that supports a direct involvement of BmPs in the final determination of digit identities in mouse embryos. Similarly, the transcriptional regulators that specify or determine the identity of a particular digit are unknown, but 5′-Hoxd and Sall genes are involved3,70.

The correct elaboration of digit identity might be assured by adjusting the PFR size and phalange shapes through local signalling interactions after the prolifera-tive expansion of the autopod primordia is completed. in fact, the PD length and number of phalanges are the defining criteria for AP digit identities, which shows how the AP and PD axes are inseparably linked. The hunt for specific genes that determine digit identity might be chasing a phantom, as identities are likely to be determined as a consequence of integrating various inputs over time and space, and definitive identities might be fixed late during chondrogenic differentia-tion. During this late phase, BmPs also participate in the differentiation and shaping of the limb skeleton and in inducing apoptosis of the interdigital mesenchyme71,72.

Limb development, evolution and malformationsin this section, we discuss the integrative model in rela-tion to normal limb bud development, the evolution of paired appendages and the causes of congenital limb mal-formations in humans and other vertebrates. The model predicts that mutations in genes that are essential for limb bud development should alter the development of both major limb bud axes, albeit with differing severity. indeed, mutations that inactivate Fg f expression in the AER not only truncate the PD axis but also affect the AP axis such that the skeletal phenotype that is caused by the loss of Fgf8 and Fgf4 is similar to that of Shh-deficient limbs9,32. in agreement, preliminary

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Box 2 | Zebrafish fin bud development and evolution

Genetic analysis of zebrafish fin buds has shown that the signalling interactions and gene networks that control fin bud development are similar to those that control tetrapod limb organogenesis in mouse and chicken embryos. Such studies have begun to show the molecular alterations that might underlie the fin to limb transition during vertebrate evolution.

The nascent fin field consists of a two- to three-cell-thick mesenchymal layer93 (see the figure, part a). Genetic evidence indicates that axial retinoic acid (RA) signalling is required for heart and neural crest derivatives 2 (Hand2) expression in the fin field94. An important step is induction of sonic hedgehog (Shh) expression in the posterior mesenchyme (part b, red) by the HAND2 and homeobox D (HOXD) transcriptional regulators (probably Hoxd10a, light blue in part b, and Hoxd11–13a, light blue dots in parts c and d)76,94. This results in the formation of a small bud owing to localized proliferation. Subsequently, an apical ectodermal ridge (AER)-like structure (part c, green) is induced by mesenchymal signals and mesenchymal fibroblast growth factor (FGF) signalling induces AER-FGF signalling and initiation of proximodistal (PD) outgrowth95. The AER is modified into the apical fold (part d, AF) as the basal ectodermal stratum folds into a double-layered epithelium.

During these stages, SHH and AER-FGF signalling propagate each other as part of an SHH–FGF epithelial–mesenchymal (e–m) feedback loop that coordinates cell proliferation (as shown by cyclin D1 (Ccnd1) expression, yellow) with anteroposterior (AP) and PD axis development76,96,97. The proximal mesenchyme condenses to give rise to cartilage (cc), and the AF together with the ingressing mesenchyme differentiates into the larval fin skeleton (by ~96 hours post fertilization (hpf))93.

A major difference between zebrafish and tetrapod paired appendage buds is the different fates of the AER, which is a transient structure in zebrafish embryos. An in-depth comparative molecular analysis of the underlying e–m signalling interactions might provide important clues about limb evolution. For example, proliferative expansion of the fin bud mesenchyme does not seem to be mediated by an SHH–FGF feedback loop, which contrasts sharply with mouse and chicken limb buds (FIGS 3,4). In the fin bud, the SHH–FGF feedback loop is likely to be terminated as the AF forms, precluding further proliferative expansion and formation of distal mesenchymal structures. Similarly, in the fin buds of fish species other than zebrafish, the distal-anterior expansion of the expression domain of 5′-located Hoxd genes (5′-Hoxd) is initiated but does not progress to the extent seen in tetrapod limb buds98,99. Prolongation of SHH–FGF feedback signalling might have enabled the evolution of distally elongated appendage structures by increasing the proliferative expansion of the mesenchymal compartment. Indeed, experimental manipulation of the length of SHH signalling in zebrafish and dogfish fin buds alters the size of the endoskeletal condensations76. ep, epidermis; LPM, lateral plate mesoderm; mes, mesenchyme; NT, neural tube; som, somite; soma, somatopleure.


Fgfs

AER

?

Anteriorc 28–31 hpf

d 37 hpf

b 23–24 hpf

Posterior

Anterior Posterior

Anterior

Dorsal

Ventral

a 12–13 hpf

Posterior

Ccnd1

Ccnd1

mes

Fgfs

ep

som

LPM

Hoxd10asoma

Shh

Shh

Shh

AF

cc

Hand2

RANT

Fgfs

Hoxd11–13a

Hoxd11–13a

results suggest that molecular markers for the PD axis are altered in Shh-deficient mouse limb buds (S. Probst and A.Z., unpublished observations).

Furthermore, the limb skeletal phenotypes caused by the inactivation of archetypal patterning genes, such as Shh, might actually arise as a consequence of altered sur-vival and/or proliferative expansion of progenitors rather than from bona fide patterning defects50. localized mes-enchymal apoptosis is the most common defect observed in mouse limb buds that lack genes such as Shh, Grem1, Bmp4 or one of the Fgf genes, and it has been difficult to correlate apoptosis patterns with the loss of particular skeletal elements8,9,14,50,55. This might be due to aberrant expansion and/or altered fates of the remaining progeni-tors. As discussed above, re-evaluation of the classical X-irradiation studies revealed that the irradiation of

developing chicken wing buds causes apoptosis of pro-liferating mesenchymal progenitors rather than alter-ing molecular markers of PD limb bud axis patterning. This localized apoptosis results in the loss of specific Sox9-positive chondrogenic elements23.

Insights into paired appendage evolution and human limb malformations. molecular analysis of zebrafish fin bud development (BOX 2) has revealed striking mecha-nistic parallels with early mouse and chicken limb bud development. For example, an SHH–FGF feedback sig-nalling loop is initiated in zebrafish fin buds, but not propagated. This suggests that the initiation and early specification phase of limb bud development are evolu-tionarily ancient. However, during fin bud development, the apical fold ‘lifts off ’ at an early stage, which suggests

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a

b Lmbr1 and Shh gene loci c Correlation between ZRS or MFCS1 and number of digits

Wild-type ZRS Five digits

Point mutations in mouse,human and cat ZRS

PPD

Deletion of the MFCS1No Shh expression in the limb bud

No hand plateOne small digit

Six toes in prehistorical rock art Infant with eight toes Hemingway cat with six toes

∗

∗

2

3

4Shh

Shh

EctopicShh

1 Mb

1 4 5 17

ShhLmbr1

ZRS

ZRSAcheiropodia deletion

21

3

4

5

5

Box 3 | Preaxial polydactylies

Digit abnormalities occur frequently in pentadactylous vertebrates (see the figure, part a), and polydactylies have intrigued humans since prehistoric times100. Polydactylies are often caused by single mutations. The most studied polydactylies — preaxial polydactylies (PPDs) — arise at a frequency of ~1 individual in 2,000 in the human population. PPD affects the anterior part of hands and/or feet, and phenotypes range from extra phalanges on thumbs to one to several additional digits (part a).

Analyses of mouse models have provided insights into molecular alterations underlying PPD and have revealed unusual features of the transcriptional regulation of the sonic hedgehog (Shh) gene in limb buds. In humans and mice with PPD, most causative point mutations map to a conserved non-coding region located in intron 5 of the limb region 1 (Lmbr1) gene101,102 (part b). The Lmbr1 locus harbours a 1.7 kb limb-specific Shh enhancer known as the zone of polarizing activity regulatory sequence (ZRS), which is located ~800 kb upstream of Shh103. Point mutations in Hemingway cats (part a) and in some cases of PPD in dogs were also mapped to the ZRS orthologous region104,105.

In the mouse PPD models, an anterior ectopic Shh expression domain is established in the embryonic limb bud (part c, middle panel)77,102. By contrast, in mice the deletion of an 800 bp region in the ZRS (mammal–fish conserved sequence 1 (MFCS1)) results in the loss of Shh expression and failure of the ulna and digits 2 to 5 to develop (part c, lower panel)106. This phenotype is identical to the Shh loss-of-function mutation in mice106. This limb phenotype also closely resembles that of human acheiropodia syndrome. The acheiropodia mutations map close to the human ZRS region107 (part b).

Understanding the long-distance regulation of Shh transcription is challenging, but a recent study establishes that the MFCS1 region physically interacts with the Shh gene by chromosomal looping108. Although it is not yet understood how the single point mutations in the ZRS cause anterior ectopic Shh expression and how MFCS1 deletion results in limb-specific loss of Shh expression, this analysis has implications for understanding the evolutionary diversification of tetrapod limbs. The genomes of some snakes and limbless amphibians lack the ZRS-MFCS1 region, which suggests that loss of this region might be causally linked to, or has paralleled, the loss of limbs77. Although early tetrapods were polydactylous4, this state has not been selected for during evolution, as digit numbers in modern tetrapods vary considerably. The relatively high incidence of PPD phenotypes in different species suggests that digit numbers are subject to fairly frequent alteration. Part a: rock art image is reproduced by permission of Rupestrian CyberServices, Flagstaff, Arizona; baby feet image Asianewsphoto; cat image is reproduced, with permission, from ReF. 104 (2008) Human Molecular Genetics.

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that e–m feedback signalling interactions are terminated early during development of the fin bud. Therefore, the proliferative expansion phase is likely to be much shorter or non-existent in fin buds compared with mouse and chicken limb buds.

comparative analysis of bat wing and leg develop-ment also supports the idea that variations in the length of the proliferative expansion phase are important for evolutionary adaptation. Specifically, a second wave of SHH and FGF signalling seems to occur in wing buds, which prolongs proliferative expansion and promotes the formation of the elongated phalanges that are char-acteristic of bat wings73. Furthermore, in lizards, short-ening of the phase of SHH signalling, possibly owing to the early termination of e–m feedback signalling, seems to reduce the number of digits in closely related species74. indeed, this reduction in digit number can be mimicked by transient, local inhibition of cell proliferation during limb bud development75.

These studies show how alterations of e–m feedback signalling and proliferative expansion might have contrib-uted to the evolution of complex fin and limb morpholo-gies in vertebrates76. One way to alter the proliferative expansion phase would be to change the temporal and/or spatial expression of the key regulatory signals. indeed, preaxial polydactyly, which is one of the most common types of congenital limb malformation in humans, is often caused by mutations in the cis-regulatory region that controls Shh expression in limb buds (BOX 3). These mutations are likely to result in the establishment of an anterior ectopic Shh expression domain, which in turn extends AER-FGF signalling. Such extended AER-FGF signalling, together with increased SHH signal transduc-tion, causes overexpansion of the autopod progenitors and results in the formation of additional digits. By con-trast, in some snakes and limbless amphibians the absence of SHH expression disrupts the initiation of outgrowth after the limb bud has formed77,78 (BOX 3). This indicates that the alterations that cause limblessness disrupt the establishment of the self-regulatory e–m feedback signal-ling system and proliferative expansion rather than limb bud formation and specification.

Conclusions and outlookRecent studies have begun to move our understanding of limb organogenesis from linear hierarchical concepts to an integrative, mechanistic view. it is becoming appar-ent that interlinked signalling feedback loops might be a hallmark of morphogenetic systems that integrate information and coordinate growth with patterning. Therefore, the next major step forward will come from deciphering how limb bud cells receive and integrate the information that controls their fate, survival and

proliferation potential. To this end, it is essential to use systems biology approaches, such as transcriptomic and proteomic analyses, in combination with mathematical modelling to look at different stages of limb develop-ment. in particular, quantitative data, space and time must all be taken into account when modelling limb organogenesis, as temporal and spatial changes in gene functions and interactions are key to the normal progres-sion of limb bud development. Therefore, it is important to develop methods that allow the visualization of path-way activity in real time — for example, by using in vivo pathway sensors in combination with four-dimensional imaging79 and quantitative methods.

more genome-wide, cellular and biochemical approaches, such as recently performed for Gli3 (ReF. 80), are urgently required to gain a deep mechanistic under-standing of how cells in the limb bud integrate informa-tion and activate the genes that define their positions and fates. For example, genetic studies in mice have just begun to reveal how lim-homeodomain tran-scription regulators integrate the signalling inputs that coordinately control limb bud patterning and growth81. Another example is that of the ETS translocation variant transcription factors ETv4 and ETv5, which participate in posterior restriction of Shh expression and are also targets of AER-FGF signalling. These two roles show that they are required for coordinated PD and AP limb bud axis development82,83.

One fascinating issue that has not been studied in detail is whether limb bud cells ‘decide’ their fate as individuals or as groups. The current evidence is sparse and contradictory: the classical morphogen gradient models propose that individual cells respond to particular thresholds, which would assign them to a particular group, as the formation of cartilage requires a large number of mesenchymal progenitors. Therefore, a particular identity could be assigned to a group of limb bud mesenchymal cells as a consequence of specific sig-nalling inputs and/or as a consequence of them belong-ing to the same group of descendants with a particular history. indeed, a mixed culture of mesenchymal cells containing cells isolated from different regions along the PD limb bud axis will sort according to where the cells originated84. light might be shed onto how and when cell and/or group fates are determined by combin-ing comprehensive cell lineage analysis with single-cell transcriptomic analysis. last, but not least, it is clear that the limb bud will continue to serve as one of the paradigmatic models of the integrative growth and patterning systems that orchestrate vertebrate develop-ment and will provide answers that are of general rel-evance to normal organogenesis and the engineering of complex tissues.

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AcknowledgementsWe are indebted to C. Müller-Thompson, J. D. Benazet, A. Galli, C. Vaillant and the three reviewers for their critical input and discussions. We wish to apologize to all our col-leagues whose important contributions could not be dis-cussed and/or referenced owing to space limitations. Our research on limb development is supported by the Swiss National Science Foundation, the European Union Marie Curie Fellowship program and the University of Basel.

DATABASESEntrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneBmp4 | Fgf4 | Fgf8 | Fgf9 | Fgf10 | Fgf17 | Gli3 | Grem1 | Hand2 | Hoxd | Lmbr1 | Shh | Sox9OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMacheiropodiapreaxial polydactylies

FURTHER INFORMATIONDevelopmental Genetics research group, University of Basel: http://biomedizin.unibas.ch/research/research-group-details/home/researchgroup/developmental-genetics

All links Are Active in the online Pdf

R E V I E W S



http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene














http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM



http://biomedizin.unibas.ch/research/research-group-details/home/researchgroup/developmental-genetics

http://biomedizin.unibas.ch/research/research-group-details/home/researchgroup/developmental-genetics

Vertebrate Limb Bud Development-moving Towards Integrative Analysis of Org a No Genesis

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