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Abstract: Chicken talpid 3 mutant embryos have a wide range of Hedgehog-signalling related defects and it is now known that the talpid 3 gene product encodes a novel protein essential for Hedgehog signalling which is required for both activator and repressor functions of Gli transcription factors (Davey, M.G., Paton, I.R., Yin, Y., Schmidt, M., Bangs, F.K., Morrice, D.R., Gordon-Smith, T., Buxton, P., Stamataki, D., Tanaka, M., Münsterberg, A.E., Briscoe, J., Tickle, C., Burt, D.W. (2006). The chicken talpid 3 gene encodes a novel protein essential for Hedgehog signalling. Genes Dev 20 1365–77). Haemorrhaging, oedema and other severe vascular defects are a central aspect of the talpid 3 phenotype (Ede, D.A. and Kelly, W.A (1964a). Developmental abnormalities in the head region of the talpid 3 mutant fowl. J. Embryol. exp. Morp. 12:161–182) and, as Hedgehog (Hh) signalling has been implicated in every stage of development of the vascular system, the vascular defects seen in talpid 3 are also likely to be attributable to abnormal Hedgehog signalling. Gene expression of members of the VEGF and Angiopoietin families of angiogenic growth factors has been linked to haemorrhaging and oedema and we find widespread expression of VEGF-D, rigf and Ang2a in the talpid 3 limb. Furthermore, ectopic expression of these genes in talpid 3 limbs points to regulation via Gli repression rather than activation. We monitored specification of vessel identity in talpid 3 limb vasculature by examining expression of artery-specific genes, Np1 and EphrinB2, and the vein-specific genes, Np2a and Tie2. We show that there are supernumerary subclavian arteries in talpid 3 limb buds and abnormal expression of an artery-specific gene in the venous submarginal sinus, despite the direction of blood flow being normal. Furthermore, we show that Shh can induce Np1 expression but has no effect on Np2a. Finally, we demonstrate that induction of VEGF and Ang2a expression by Shh in normal limb buds is accompanied by vascular remodelling. Thus Hedgehog signalling has a pivotal role in the cascade of angiogenic events in a growing embryonic organ which is similar to that proposed in tumours. [Copyright &y& Elsevier]

The chick embryo has a long history in investigations of vertebrate limb development because of the ease with which its limbs can be experimentally manipulated. Early studies elucidated the fundamental embryology of the limb and identified the key signalling regions that govern its development. The chick limb became a leading model for exploring the concept of positional information and understanding how patterns of differentiated cells and tissues develop in vertebrate embryos. When developmentally important molecules began to be identified, experiments in chick limbs were crucial for bridging embryology and molecular biology. The embryological mechanisms and molecular basis of limb development are largely conserved in mammals, including humans, and uncovering these molecular networks provides links to clinical genetics. We emphasise the important contributions of naturally occurring chick mutants to elucidating limb embryology and identifying novel developmentally important genes. In addition, we consider how the chick limb has been used to study mechanisms involved in teratogenesis with a focus on thalidomide. These studies on chick embryos have given insights into how limb defects can be caused by both genetic changes and chemical insults and therefore are of great medical significance.

John Saunders was a highly skilled embryologist who pioneered the study of limb development. His studies on chick embryos provided the fundamental framework for understanding how vertebrate limbs develop. This framework inspired generations of scientists and formed the bridge from experimental embryology to molecular mechanisms. Saunders investigated how feathers become organized into tracts in the skin of the chick wing and also identified regions of programmed cell death. He discovered that a region of thickened ectoderm that rims the chick wing bud - the apical ectodermal ridge - is required for outgrowth and the laying down of structures along the proximo-distal axis (long axis) of the wing, identified the zone of polarizing activity (ZPA; polarizing region) that controls development across the anteroposterior axis ("thumb to little finger "axis) and contributed to uncovering the importance of the ectoderm in development of structures along the dorso-ventral axis ( "back of hand to palm" axis). This review looks in depth at some of his original papers and traces how he made the crucial findings about how limbs develop, considering these findings both in the context of contemporary knowledge at the time and also in terms of their immediate impact on the field., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The gene encoding the secreted protein Sonic hedgehog (Shh) is expressed in the polarizing region (or zone of polarizing activity), a small group of mesenchyme cells at the posterior margin of the vertebrate limb bud. Detailed analyses have revealed that Shh has the properties of the long sought after polarizing region morphogen that specifies positional values across the antero-posterior axis (e.g., thumb to little finger axis) of the limb. Shh has also been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation and by regulating the antero-posterior length of the apical ectodermal ridge, the signaling region required for limb bud outgrowth and the laying down of structures along the proximo-distal axis (e.g., shoulder to digits axis) of the limb. It has been shown that Shh signaling can specify antero-posterior positional values in limb buds in both a concentration- (paracrine) and time-dependent (autocrine) fashion. Currently there are several models for how Shh specifies positional values over time in the limb buds of chick and mouse embryos and how this is integrated with growth. Extensive work has elucidated downstream transcriptional targets of Shh signaling. Nevertheless, it remains unclear how antero-posterior positional values are encoded and then interpreted to give the particular structure appropriate to that position, for example, the type of digit. A distant cis-regulatory enhancer controls limb-bud-specific expression of Shh and the discovery of increasing numbers of interacting transcription factors indicate complex spatiotemporal regulation. Altered Shh signaling is implicated in clinical conditions with congenital limb defects and in the evolution of the morphological diversity of vertebrate limbs.

A fundamental question in biology is how the extraordinary range of living organisms arose. In this theme issue, we celebrate how evolutionary studies on the origins of morphological diversity have changed over the past 350 years since the first publication of the Philosophical Transactions of The Royal Society Current understanding of this topic is enriched by many disciplines, including anatomy, palaeontology, developmental biology, genetics and genomics. Development is central because it is the means by which genetic information of an organism is translated into morphology. The discovery of the genetic basis of development has revealed how changes in form can be inherited, leading to the emergence of the field known as evolutionary developmental biology (evo-devo). Recent approaches include imaging, quantitative morphometrics and, in particular, genomics, which brings a new dimension. Articles in this issue illustrate the contemporary evo-devo field by considering general principles emerging from genomics and how this and other approaches are applied to specific questions about the evolution of major transitions and innovations in morphology, diversification and modification of structures, intraspecific morphological variation and developmental plasticity. Current approaches enable a much broader range of organisms to be studied, thus building a better appreciation of the origins of morphological diversity.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'., (© 2016 The Author(s).)

Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development.

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors (FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein (BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs, respectively, the apical ectodermal ridge producing fibroblast growth factors and the polarizing region, Sonic hedgehog (Shh). These signals are the same in both wings/forelimbs and legs/hindlimbs and control growth and pattern formation by providing the mesoderm cells of the limb bud as it develops with positional information. The precise anatomy of the limb depends on the mesoderm cells in the developing bud interpreting positional information according to their identity - determined by Pitx1 in hindlimbs - and genotype. The competence to form a limb extends along the entire antero-posterior axis of the trunk - with Hox gene activity inhibiting the formation of forelimbs in the interlimb region - and also along the dorso-ventral axis., (© 2015 Anatomical Society.)

MicroRNAs are differentially expressed in breast cancer cells and have been implicated in cancer formation, tumour invasion and metastasis. We investigated the miRNA expression profiles in the developing mammary gland. MiR-137 was expressed prominently in the developing mammary gland. When the miR-137 was over-expressed in the embryo, the mammary epithelium became thickened. Moreover, genes associated with mammary gland formation such as Tbx3 and Lef1 were not expressed. This suggests that miR-137 induces gland formation and invasion. When miR-137 was over-expressed in MDA-MB-231 cells, their ability to form tumours in adult mice was significantly reduced. These data support miR-137 decides epithelial cell behavior in the human breast cancer. It also suggests that miR-137 is a potential therapeutic target for amelioration of breast cancer progression.

How time is measured is an enduring issue in developmental biology. Classical models of somitogenesis and limb development implicated intrinsic cell cycle clocks, but their existence remains controversial. Here we show that an intrinsic cell cycle clock in polarizing region cells of the chick limb bud times the duration of Sonic hedgehog (Shh) expression, which encodes the morphogen specifying digit pattern across the antero-posterior axis (thumb to little finger). Timing by this clock starts when polarizing region cells fall out of range of retinoic acid signalling. We found that timing of Shh transcription by the cell cycle clock can be reset, thus revealing an embryonic form of self-renewal. In contrast, antero-posterior positional values cannot be reset, suggesting that this may be an important constraint on digit regeneration. Our findings provide the first evidence for an intrinsic cell cycle timer controlling duration and patterning activity of a major embryonic signalling centre.

Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. Here, we show that MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. Our data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73., (© 2014. Published by The Company of Biologists Ltd.)

Background: The three chick wing digits represent a classical example of a pattern specified by a morphogen gradient. Here we have investigated whether a mathematical model of a Shh gradient can describe the specification of the identities of the three chick wing digits and if it can be applied to limbs with more digits., Results: We have produced a mathematical model for specification of chick wing digit identities by a Shh gradient that can be extended to the four digits of the chick leg with Shh-producing cells forming a digit. This model cannot be extended to specify the five digits of the mouse limb., Conclusions: Our data suggest that the parameters of a classical-type morphogen gradient are sufficient to specify the identities of three different digits. However, to specify more digit identities, this core mechanism has to be coupled to alternative processes, one being that in the chick leg and mouse limb, Shh-producing cells give rise to digits; another that in the mouse limb, the cellular response to the Shh gradient adapts over time so that digit specification does not depend simply on Shh concentration., (Copyright © 2013 Wiley Periodicals, Inc.)

A gradient of Sonic hedgehog (Shh) plays a major role in specifying the antero-posterior pattern of structures that develop in the distal part of the vertebrate limb, in particular, the antero-posterior pattern of the digits. Classical embryological experiments identified the polarizing region (or zone of polarizing activity, ZPA), a signaling region at the posterior margin of the early chick wing bud and, consistent with a model in which production of a diffusible morphogen specifies antero-posterior positional information, polarizing region signaling was shown to be dose dependent and long range. It is now well established that the vertebrate hedgehog gene, Sonic hedgehog (Shh), which encodes a secreted protein, is expressed in the polarizing region of the chick wing and that Shh signaling has the same characteristics as polarizing region signaling. Shh expression at the posterior of the early limb bud and the mechanism of Shh signal transduction are conserved among vertebrates including mammals. However, it is unlikely that a simple Shh gradient is responsible for digit pattern formation in mammalian limbs and there is still little understanding of how positional information specified by Shh signaling is encoded and translated into digit anatomy. Alterations in Shh signaling underlie some congenital limb abnormalities and also changes in timing and extent of Shh signaling appear to be related to the evolution of morphological diversity of vertebrate limbs., (Copyright © 2012 Wiley Periodicals, Inc.)

Retinoic acid receptors (RARs), which are involved in retinoic acid signal transduction, are essential for maintaining the differentiated state of epithelial tissues. Mammary glands are skin appendages whose development is initiated through continuous cell-cell interactions between the ectoderm and the adjacent mesenchyme. Considerable progress has been made in elucidating the molecular basis of these interactions in mammary gland formation in mouse embryos, including the network of initiating signals comprising Fgfs, Wnts and Bmps involved in gland positioning and the transcription factors, Tbx3 and Lef1, essential for mammary gland development. Here, we provide evidence that retinoic acid signaling may also be involved in mammary gland development. We documented the expression of gene-encoding enzymes that produce retinoic acid (Raldh2) and enzymes that degrade it (Cyp26a1, Cyp26b1). We also analyzed the expression of RAR-β, a direct transcriptional target of retinoic acid signaling. Raldh2 and RAR-β were expressed in E10-E10.5 mouse embryos in somites adjacent to the flank region where mammary buds 2, 3 and 4 develop. These expression patterns overlapped with that of Fgf10, which is known to be required for mammary gland formation. RAR-β was also expressed in the mammary mesenchyme in E12 mouse embryos; RAR-β protein was expressed in the mammary epithelium and developing fat pad. Retinoic acid levels in organ cultures of E10.5 mouse embryo flanks were manipulated by adding either retinoic acid or citral, a retinoic acid synthesis inhibitor. Reduced retinoic acid synthesis altered the expression of genes involved in retinoic acid homeostasis and also demonstrated that retinoic acid signaling is required for Tbx3 expression, whereas high levels of retinoic acid signaling inhibited Bmp4 expression and repressed Wnt signaling. The results of the experiments using RNAi against Tbx3 and Wnt10b suggested feedback interactions that regulate retinoic acid homeostasis in mammary gland-forming regions. We produced a molecular model for mammary gland initiation that incorporated retinoic acid signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The developing limb is one of the first systems where it was proposed that a signalling gradient is involved in pattern formation. This gradient for specifying positional information across the antero-posterior axis is based on Sonic hedgehog signalling from the polarizing region. Recent evidence suggests that Sonic hedgehog signalling also specifies positional information across the antero-posterior axis by a timing mechanism acting in parallel with graded signalling. The progress zone model for specifying proximo-distal pattern, involving timing to provide cells with positional information, continues to be challenged, and there is further evidence that graded signalling by retinoic acid specifies the proximal part of the limb. Other recent papers present the first evidence that gradients of signalling by Wnt5a and FGFs govern cell behaviour involved in outgrowth and morphogenesis of the developing limb., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region. To determine how digit identity is specified over time, we inhibited Sonic hedgehog signalling. Fate maps show that polarizing region and adjacent cells are specified in parallel through a series of anterior to posterior digit fates-a process of digit specification that we suggest is involved in patterning all vertebrate limbs with more than three digits.

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.

Sonic hedgehog (Shh) signalling controls integrated specification of digit pattern and growth in the chick wing but downstream gene networks remain to be unravelled. We analysed 3D expression patterns of genes encoding cell cycle regulators using Optical Projection Tomography. Hierarchical clustering of spatial matrices of gene expression revealed a dorsal layer of the wing bud, in which almost all genes were expressed, and that genes encoding positive cell cycle regulators had similar expression patterns while those of N-myc and CyclinD2 were distinct but closely related. We compared these patterns computationally with those of genes implicated in digit specification and Ptch1, 50 genes in total. Nineteen genes have similar posterior expression to Ptch1, including Hoxd13, Sall1, Hoxd11, and Bmp2, all likely Gli targets in mouse limb, and cell cycle genes, N-myc, CyclinD2. We suggest that these genes contribute to a network integrating digit specification and growth in response to Shh., (Copyright © 2011 Wiley-Liss, Inc.)

Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly., (Copyright © 2011 Wiley-Liss, Inc.)

Each vertebrate species displays specific tooth patterns in each quadrant of the jaw: the mouse has one incisor and three molars, which develop at precise locations and at different times. The reason why multiple teeth form in the jaw of vertebrates and the way in which they develop separately from each other have been extensively studied, but the genetic mechanism governing the spatial patterning of teeth still remains to be elucidated. Sonic hedgehog (Shh) is one of the key signaling molecules involved in the spatial patterning of teeth and other ectodermal organs such as hair, vibrissae and feathers. Sostdc1, a secreted inhibitor of the Wnt and Bmp pathways, also regulates the spatial patterning of teeth and hair. Here, by utilizing maternal transfer of 5E1 (an anti-Shh antibody) to mouse embryos through the placenta, we show that Sostdc1 is downstream of Shh signaling and suggest a Wnt-Shh-Sostdc1 negative feedback loop as a pivotal mechanism controlling the spatial patterning of teeth. Furthermore, we propose a new reaction-diffusion model in which Wnt, Shh and Sostdc1 act as the activator, mediator and inhibitor, respectively, and confirm that such interactions can generate the tooth pattern of a wild-type mouse and can explain the various tooth patterns produced experimentally.

Hoxd13, Tbx2, Tbx3, Sall1 and Sall3 genes are candidates for encoding antero-posterior positional values in the developing chick wing and specifying digit identity. In order to build up a detailed profile of gene expression patterns in cell lineages that give rise to each of the digits over time, we compared 3 dimensional (3D) expression patterns of these genes during wing development and related them to digit fate maps. 3D gene expression data at stages 21, 24 and 27 spanning early bud to digital plate formation, captured from in situ hybridisation whole mounts using Optical Projection Tomography (OPT) were mapped to reference wing bud models. Grafts of wing bud tissue from GFP chicken embryos were used to fate map regions of the wing bud giving rise to each digit; 3D images of the grafts were captured using OPT and mapped on to the same models. Computational analysis of the combined computerised data revealed that Tbx2 and Tbx3 are expressed in digit 3 and 4 progenitors at all stages, consistent with encoding stable antero-posterior positional values established in the early bud; Hoxd13 and Sall1 expression is more dynamic, being associated with posterior digit 3 and 4 progenitors in the early bud but later becoming associated with anterior digit 2 progenitors in the digital plate. Sox9 expression in digit condensations lies within domains of digit progenitors defined by fate mapping; digit 3 condensations express Hoxd13 and Sall1, digit 4 condensations Hoxd13, Tbx3 and to a lesser extent Tbx2. Sall3 is only transiently expressed in digit 3 progenitors at stage 24 together with Sall1 and Hoxd13; then becomes excluded from the digital plate. These dynamic patterns of expression suggest that these genes may play different roles in digit identity either together or in combination at different stages including the digit condensation stage.

Eggs containing live Japanese quail embryos were imaged using micro-magnetic resonance imaging (μMRI) at 24-h intervals from Day 0 to 8, the period during which the main body axis is being laid down and organogenesis is taking place. Considerable detail of non-embryonic structures such as the latebra was revealed at early stages but the embryo could only be visualized around Day 3. Three-dimensional (3D) changes in embryo length and volume were quantified and also changes in volume in the extra- and non-embryonic components. The embryo increased in length by 43% and nearly trebled in volume between Day 4 and Day 5. Although the amount of yolk remained fairly constant over the first 5 days, the amount of albumen decreases significantly and was replaced by extra-embryonic fluid (EEF). ¹H longitudinal (T₁) and transverse (T₂) relaxation times of different regions within the eggs were determined over the first 6 days of development. The T₂ measurements mirrored the changes in image intensity observed, which can be related to the aqueous protein concentrations. In addition, a comparison of the development of Day 0 to 3 quail embryos exposed to radiofrequency (rf) pulses, 7 T static magnetic fields and magnetic field gradients for an average of 7 h with the development of control embryos did not reveal any gross changes, thus confirming that μMRI is a suitable tool for following the development of live avian embryos over time from the earliest stages., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sonic hedgehog (Shh) signalling by the polarizing region at the posterior margin of the chick wing bud is pivotal in patterning the digits but apart from a few key downstream genes, such as Hoxd13, which is expressed in the posterior region of the wing that gives rise to the digits, the genes that mediate the response to Shh signalling are not known. To find genes that are co-expressed with Hoxd13 in the posterior of chick wing buds and regulated in the same way, we used microarrays to compare gene expression between anterior and posterior thirds of wing buds from normal chick embryos and from polydactylous talpid³ mutant chick embryos, which have defective Shh signalling due to lack of primary cilia. We identified 1070 differentially expressed gene transcripts, which were then clustered. Two clusters contained genes predominantly expressed in posterior thirds of normal wing buds; in one cluster, genes including Hoxd13, were expressed at high levels in anterior and posterior thirds in talpid³ wing buds, in the other cluster, genes including Ptc1, were expressed at low levels in anterior and posterior thirds in talpid³ wing buds. Expression patterns of genes in these two clusters were validated in normal and talpid³ mutant wing buds by in situ hybridisation and demonstrated to be responsive to application of Shh. Expression of several genes in the Hoxd13 cluster was also shown to be responsive to manipulation of protein kinase A (PKA) activity, thus demonstrating regulation by Gli repression. Genes in the Hoxd13 cluster were then sub-clustered by computational comparison of 3D expression patterns in normal wing buds to produce syn-expression groups. Hoxd13 and Sall1 are syn-expressed in the posterior region of early chick wing buds together with 6 novel genes which are likely to be functionally related and represent secondary targets of Shh signalling. Other groups of syn-expressed genes were also identified, including a group of genes involved in vascularisation., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In Drosophila, Iroquois (Irx) genes have various functions including the specification of the identity of wing veins. Vertebrate Iroquois (Irx) genes have been reported to be expressed in the developing digits of mouse limbs. Here we carry out a phylogenetic analysis of vertebrate Irx genes and compare expression in developing limbs of mouse, chick and human embryos and in zebrafish pectoral fin buds. We confirm that the six Irx gene families in vertebrates are well defined and that Clusters A and B are duplicates; in contrast, Irx1 and 3, Irx2 and 5, and Irx4 and 6 are paralogs. All Irx genes in mouse and chick are expressed in developing limbs. Detailed comparison of the expression patterns in mouse and chick shows that expression patterns of genes in the same cluster are generally similar but paralogous genes have different expression patterns. Mouse and chick Irx1 are expressed in digit condensations, whereas mouse and chick Irx6 are expressed interdigitally. The timing of Irx1 expression in individual digits in mouse and chick is different. Irx1 is also expressed in digit condensations in developing human limbs, thus showing conservation of expression of this gene in higher vertebrates. In zebrafish, Irx genes of all but six of the families are expressed in early stage pectoral fin buds but not at later stages, suggesting that these genes are not involved in patterning distal structures in zebrafish fins.

Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. SHOX gene mutations and deletions of regulatory elements downstream of SHOX resulting in haploinsufficiency have been found in patients with LWD. SHOX encodes a homeodomain transcription factor and is known to be expressed in the developing limb. We have now analyzed the regulatory significance of the region upstream of the SHOX gene. By comparative genomic analyses, we identified several conserved non-coding elements, which subsequently were tested in an in ovo enhancer assay in both chicken limb bud and cornea, where SHOX is also expressed. In this assay, we found three enhancers to be active in the developing chicken limb, but none were functional in the developing cornea. A screening of 60 LWD patients with an intact SHOX coding and downstream region did not yield any deletion of the upstream enhancer region. Thus, we speculate that SHOX upstream deletions occur at a lower frequency because of the structural organization of this genomic region and/or that SHOX upstream deletions may cause a phenotype that differs from the one observed in LWD.

Gross similarities between the external appearance of the hind limbs of the peroneal muscle atrophy (pma) mouse mutant and congenital talipes equinovarus (CTEV), a human disorder historically referred to as 'clubfoot', suggested that this mutant could be a useful model. We used micro-magnetic resonance imaging to visualize the detailed anatomy of the hind limb defect in mutant pma mice and performed 3D comparisons between mutant and wild-type hind limbs. We found that the pma foot demonstrates supination (i.e. adduction and inversion of the mid foot and fore foot together with plantar flexion of the ankle and toes) and that the tibiale and distal tarsals display 3D abnormalities in positioning. The size and shape of the tibia, fibula, tarsal and metatarsal bones are similar to the wild-type. Hypoplasia of the muscles in the antero-lateral (peroneal) compartment was also demonstrated. The resemblance of these features to those seen in CTEV suggests that the pma mouse is a possibly useful model for the human condition. To understand how the observed deformities in the pma mouse hind foot arise during embryonic development, we followed the process of foot rotation in both wild-type and pma mutant mice. Rotation of the hind foot in mouse embryos of wild-type strains (CD-1 and C57/Black) occurs from embryonic day 14.5 onwards with rotation in C57/Black taking longer. In embryos from both strains, rotation of the right hind foot more commonly precedes rotation of the left. In pma mutants, the initiation of rotation is often delayed and rotation is slower and does not reach completion. If the usefulness of the pma mutant as a model is confirmed, then these findings on pma mouse embryos, when extrapolated to humans, would support a long-standing hypothesis that CTEV is due to the failure of completion of the normal process of rotation and angulation, historically known as the 'arrested development hypothesis'.

The E2F family of transcriptional regulators activate or repress gene expression during specific phases of the cell cycle and control various processes including proliferation, apoptosis and differentiation. However, little is known about the developmental roles of E2F transcription factors in higher vertebrates. The chick wing is an excellent system for studying these processes because, in addition to having a rich classical embryology, it is increasingly amenable to molecular and genomic approaches. We show that the human and mouse complement of eight E2F transcription factors is conserved in the chicken and that chicken E2F genes are expressed in different spatial and temporal patterns during wing development. We discuss how the expression patterns of the eight chicken E2F transcription factors might be related to important morphogenetic events.

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

The chicken talpid(3) mutant, with polydactyly and defects in other embryonic regions that depend on hedgehog (Hh) signalling (e.g. the neural tube), has a mutation in KIAA0568. Similar phenotypes are seen in mice and in human syndromes with mutations in genes that encode centrosomal or intraflagella transport proteins. Such mutations lead to defects in primary cilia, sites where Hh signalling occurs. Here, we show that cells of talpid(3) mutant embryos lack primary cilia and that primary cilia can be rescued with constructs encoding Talpid3. talpid(3) mutant embryos also develop polycystic kidneys, consistent with widespread failure of ciliogenesis. Ultrastructural studies of talpid(3) mutant neural tube show that basal bodies mature but fail to dock with the apical cell membrane, are misorientated and almost completely lack ciliary axonemes. We also detected marked changes in actin organisation in talpid(3) mutant cells, which may explain misorientation of basal bodies. KIAA0586 was identified in the human centrosomal proteome and, using an antibody against chicken Talpid3, we detected Talpid3 in the centrosome of wild-type chicken cells but not in mutant cells. Cloning and bioinformatic analysis of the Talpid3 homolog from the sea anemone Nematostella vectensis identified a highly conserved region in the Talpid3 protein, including a predicted coiled-coil domain. We show that this region is required to rescue primary cilia formation and neural tube patterning in talpid(3) mutant embryos, and is sufficient for centrosomal localisation. Thus, Talpid3 is one of a growing number of centrosomal proteins that affect both ciliogenesis and Hh signalling.

The developing limb has been a very influential system for studying pattern formation in vertebrates. In the past, classical embryological models have explained how patterned structures are generated along the two principal axes of the limb: the proximodistal (shoulder to finger) and anteroposterior (thumb to little finger) axes. Over time, the genetic and molecular attributes of these patterning models have been discovered, while the role of growth in the patterning process has been only recently highlighted. In this review, we discuss these recent findings and propose how the various models of limb patterning can be reconciled.

The developing limb is a major model for pattern formation in vertebrate embryos. Many of the seminal discoveries of the mechanisms involved in patterning have been made using chick embryos because of the ease of manipulating their developing limbs. More recently, the molecular basis of limb pattern formation has been increasingly uncovered and now, with the availability of genomic resources, the genetic approaches available are even more powerful. Nevertheless, since the limb is ultimately built of cells, gene action must ultimately be translated into cell behaviour and a major challenge will be to integrate genetics with molecular and cellular biology. In this review, we will first outline the stages in limb development, the major interacting signalling pathways that pattern the limb and the molecules involved. We will describe fate maps of the developing limb, and discuss what is known about cellular activities including proliferation, death, adhesiveness, communication and migration during the patterning process. Finally we will explore how these cell activities produce form.

Chick embryos are good models for vertebrate development due to their accessibility and manipulability. Recent large increases in available genomic data from both whole genome sequencing and EST projects provide opportunities for identifying many new developmentally important chicken genes. Traditional methods of documenting when and where specific genes are expressed in embryos using whole amount and section in-situ hybridisation do not readily allow appreciation of 3-dimensional (3D) patterns of expression, but this can be accomplished by the recently developed microscopy technique, Optical Projection Tomography (OPT). Here we show that OPT data on the developing chick wing from different labs can be reliably integrated into a common database, that OPT is efficient in capturing 3D gene expression domains and that such domains can be meaningfully compared. Novel protocols are used to compare 3D expression domains of 7 genes known to be involved in chick wing development. This reveals previously unappreciated relationships and demonstrates the potential, using modern genomic resources, for building a large scale 3D atlas of gene expression. Such an atlas could be extended to include other types of data, such as fate maps, and the approach is also more generally applicable to embryos, organs and tissues.

In the classical model of chick wing digit-patterning, the polarizing region--a group of cells at the posterior margin of the early bud--produces a morphogen gradient, now known to be based on Sonic hedgehog (Shh), that progressively specifies anteroposterior positional identities in the posterior digit-forming region. Here we add an integral growth component to this model by showing that Shh-dependent proliferation of prospective digit progenitor cells is essential for specifying the complete pattern of digits across the anteroposterior axis. Inhibiting Shh signalling in early wing buds reduced anteroposterior expansion, and posterior digits were lost because all prospective digit precursors formed anterior structures. Inhibiting proliferation also irreversibly reduced anteroposterior expansion, but instead anterior digits were lost because all prospective digit precursors formed posterior structures. When proliferation recovered in such wings, Shh transcription was maintained for longer than normal, suggesting that duration of Shh expression is controlled by a mechanism that measures proliferation. Rescue experiments confirmed that Shh-dependent proliferation controls digit number during a discrete time-window in which Shh-dependent specification normally occurs. Our findings that Shh signalling has dual functions that can be temporally uncoupled have implications for understanding congenital and evolutionary digit reductions.

Chick embryos are useful models for probing developmental mechanisms including those involved in organogenesis. In addition to classic embryological manipulations, it is possible to test the function of molecules and genes while the embryo remains within the egg. Here we define conditions for imaging chick embryo anatomy and for visualising living quail embryos. We focus on the developing limb and describe how different tissues can be imaged using micro-magnetic resonance imaging and this information then synthesised, using a three-dimensional visualisation package, into detailed anatomy. We illustrate the potential for micro-magnetic resonance imaging to analyse phenotypic changes following chick limb manipulation. The work with the living quail embryos lays the foundations for using micro-magnetic resonance imaging as an experimental tool to follow the consequences of such manipulations over time.

Sonic Hedgehog (Shh) signaling by the polarizing region, at the posterior of the vertebrate limb bud, is pivotal in determining digit number and identity. Shh establishes a gradient of the bifunctional transcriptional effector, Gli3, with high levels of full-length activator (Gli3A) in the posterior bud, where digits form, and high levels of shorter repressor (Gli3R) in the anterior. Repressor formation depends on protein kinase A (PKA), but in Drosophila, PKA also plays a role in activator function. Increasing PKA levels in chick limb development using Forskolin had no effect on posterior polarizing activity but weak polarizing activity, based on ligand-independent Shh signaling, was induced in anterior limb bud cells resulting in extra digits. Manipulating PKA activity levels directly with a retrovirus expressing activated PKA induced extra digits similar to those induced by Forskolin treatment suggesting that PKA may have a previously unrecognized positive role in Shh signaling in vertebrate limbs. Expressing dominant negative PKA also induced extra, sometimes multiple digits, from anterior limb bud demonstrating the negative role in Shh signaling. PKA levels in the limb bud are high posteriorly and low anteriorly, suggesting that PKA activity may influence the outcome of Shh signaling in normal development.