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.
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.
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’.
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.
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.
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.
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.
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
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.
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 evolution of snakes involved major changes in vertebrate body plan organization, but the developmental basis of those changes is unknown. The python axial skeleton consists of hundreds of similar ...vertebrae, forelimbs are absent and hindlimbs are severely reduced.
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