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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.)

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.

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'.

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.

Interactions between Wnts, Fgfs and Tbx genes are involved in limb initiation and the same gene families have been implicated in mammary gland development. Here we explore how these genes act together in mammary gland initiation. We compared expression of Tbx3, the gene associated with the human condition ulnar-mammary syndrome, expression of the gene encoding the dual-specificity MAPK phosphatase Pyst1/MKP3, which is an early response to FGFR1 signalling (as judged by sensitivity to the SU5402 inhibitor), and expression of Lef1, encoding a transcription factor mediating Wnt signalling and the earliest gene so far known to be expressed in mammary gland development. We found that Tbx3 is expressed earlier than Lef1 and that Pyst1 is also expressed early but only transiently. Patterns of expression of Tbx3, Pyst1 and Lef1 in different glands suggest that the order of mammary gland initiation is 3, 4, 1, 2 and 5. Consistent with expression of Pyst1 in the mammary gland, we detected expression of Fgfr1b, Fgf8 and Fgf9 in both surface ectoderm and mammary bud epithelium, and Fgf4 and Fgf17 in mammary bud epithelium. Beads soaked in FGF-8 applied to the flank of mouse embryos, at a stage just prior to mammary bud initiation, induce expression of Pyst1 and Lef1 and maintain Tbx3 expression in flank tissue surrounding the bead. Grafting beads soaked in the FGFR1 inhibitor, SU5402, abolishes Tbx3, Pyst1 and Lef1 expression, supporting the idea that FGFR1 signalling is required for early mammary gland initiation. We also showed that blocking Wnt signalling abolishes Tbx3 expression but not Pyst1 expression. These data, taken together with previous findings, suggest a model in which Tbx3 expression is induced and maintained in early gland initiation by both Wnt and Fgf signalling through FGFR1.

A human polydactylous left foot with 9 toes, amputated from an 11-mo-old child, was examined by x-ray and magnetic resonance imaging and by gross dissection to identify the digits. The normal sequence of toes from medial to lateral is 1, 2, 3, 4, 5. Examination of the morphology of tendons and muscles suggested the toe sequence was 1, 2, 3/4, ?5, 2, 3/4, 3/4, 5. The 2 toes in the sequence that are underlined were displaced dorsally and were found to have 2 extensor tendons, no flexor tendons and nails that were conical and situated at their tips. These toes resembled those described as 'double-dorsal' and which develop in paws of mice in which a gene normally expressed ventrally is functionally inactivated (Loomis et al. 1996). Specification of toe formation occurs in leg buds early in embryonic development and later there is rotation of the limb so that the anterior (rostral) part comes to lie medially, i.e. the hallux which was anterior (rostral) now is on the inner (medial) side of the foot. A disruption in the patterning of this foot in both anteroposterior (rostral-caudal) and dorsoventral axes during development could be responsible.