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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.017 Byline: M.G. Davey (a)(c), J. James (b), I.R. Paton (c), D.W. Burt (c), C. Tickle (a) Keywords: talpid.sup.3 ; Hedgehog; Limb; Vascular development; VEGF; Angiopoietin Abstract: Chicken talpid.sup.3 mutant embryos have a wide range of Hedgehog-signalling related defects and it is now known that the talpid.sup.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., Munsterberg, A.E., Briscoe, J., Tickle, C., Burt, D.W. (2006). The chicken talpid.sup.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.sup.3 phenotype (Ede, D.A. and Kelly, W.A (1964a). Developmental abnormalities in the head region of the talpid.sup.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.sup.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.sup.3 limb. Furthermore, ectopic expression of these genes in talpid.sup.3 limbs points to regulation via Gli repression rather than activation. We monitored specification of vessel identity in talpid.sup.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.sup.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. Author Affiliation: (a) Division of Cell and Developmental Biology, WTB, University of Dundee, Dundee, DD1 5EH, UK (b) CHIPs, WTB, University of Dundee, Dundee, DD1 5EH, UK (c) Division of Genetics and Genomics, Roslin Institute, Roslin Biocentre, Midlothian, EH25 9PS, UK Article History: Received 31 March 2006; Revised 19 July 2006; Accepted 4 August 2006

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

Bone morphogenetic proteins (BMPs) induce ectopic cartilage and bone when implanted intramuscularly in adult rats. Expression data suggest that BMPs signal skeletal development in embryos. An important question is which cells are targets of BMP signaling in adult and embryonic tissues. Here, we examined the effect of BMP-2 on micromass cultures of chick limb bud mesenchyme. We report that BMP-2 promotes formation of cartilage and simultaneously inhibits development of muscle cells. To follow the fate of presumptive muscle cells, we replaced chick somites with quail somites at the wing level and made micromass cultures from the chimeric wings. In untreated cultures, quail cells formed muscle but not cartilage. In BMP-2-treated cultures, quail cells disappeared altogether. This suggests that BMP-2 may simultaneously promote cartilage differentiation and reduce the presumptive myogenic cell populations in regions of skeletal development.

Application of a site-specific polyclonal antibody and confocal microscopy formed part of a study of the pattern of connexin43 expression in developing chick limb buds. Phases of limb growth, where epithelial mesenchymal interactions mediate morphogenesis, exhibit connexin 43 expression. These epithelial mesenchymal interactions are associated with the synthesis of this gap junction protein. This is evident as termination of bud outgrowth results in elimination of connexin43 expression in mesenchymal and epithelial domains.

Major changes in body plan organization occurred during evolution of snakes, but the developmental basis of these changes is unknown. In pythons, the axial skeleton is comprised of hundreds of morphologically similar vertebrae, forelimbs are absent and hindlimbs are severely reduced. In order to identify developmental mechanisms underlying evolution of limblessness and vertebral pattern in snakes, we have undertaken a molecular analysis of limb development and axial regionalization in python embryos, and compared the results to an identical analysis of chick embryos, a well-characterized model for tetrapod development. During chick development, paired limb buds are induced at specific locations along the body axis by local production of fibroblast growth factors. After limb bud initiation, continued outgrowth and patterning depends on a complex set of molecular interactions that coordinates proximodistal, dorsoventral & anteroposterior axes. Disruption of these interactions in chick limb buds can lead to pattern alterations or limb truncation. Our comparative analysis of gene expression, together with experimental manipulations, has identified a breakdown in this genetic circuit in python limb buds, which accounts for the early arrest of python limb development. The results suggest distinct developmental mechanisms for the major morphological transitions in snake evolution.

The traditional strength of chicken embryos for studying development is that they are readily manipulated. This has led to some major discoveries in developmental biology such as the demonstration that the neural crest gives rise to almost the entire peripheral nervous system and the identification of signalling centres that specify the pattern of structures in the central nervous system and limb. More recently with the burgeoning discovery of developmentally important genes, chicken embryos have provided useful models for testing function. Uncovering the molecular basis of development provides direct links with clinical genetics. In addition, since many genes that have crucial roles in development are also expressed in tumours, basic research on chickens has implications for understanding human health and disease. Now that the chicken genome has been sequenced and genomic resources for chicken are becoming increasingly available, this opens up opportunities for combining these new technologies with the manipulability of chicken embryos and also exploiting comparative genomics. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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

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

Abstract A detailed and precise picture is being pieced together about how the pattern of digits develops in vertebrate limbs. What is particularly exciting is that it will soon be possible to trace the process all the way from establishment of a signalling centre in a small bud of undifferentiated cells right through to final limb anatomy. The development of the vertebrate limb is a traditional model in which to explore mechanisms involved in pattern formation, and there is accelerating knowledge about the genes involved. One reason why the limb is holding its place in the post-genomic age is that it is rich in pre-genomic embryology. Here, we will focus on recent findings about the aspect of vertebrate limb development concerned with digit pattern across the antero-posterior axis of the limb. This process is controlled by a signalling region in the early limb bud known as the polarizing region. Interactions between polarizing region cells and other cells in the limb bud ensure that a thumb develops at one edge of the hand (anterior) and a little finger at the other (posterior). [ABSTRACT FROM AUTHOR]

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.)