Malcolm P. O. Logan

Tbx4 and Tbx5 are two closely related T-box genes that encode transcription factors expressed in the prospective hindlimb and forelimb territories, respectively, of all jawed vertebrates. Despite their striking limb type-restricted expression pattern, we have shown that these genes do not participate in the acquisition of limb type-specific morphologies. Instead, Tbx4 and Tbx5 play similar roles in the initiation of hindlimb and forelimb outgrowth, respectively. We hypothesized that different combinations of Hox proteins expressed in different rostral and caudal domains of the lateral plate mesoderm, where limb induction occurs, might be involved in regulating the limb type-restricted expression of Tbx4 and Tbx5 and in the later determination of limb type-specific morphologies. Here, we identify the minimal regulatory element sufficient for the earliest forelimb-restricted expression of the mouse Tbx5 gene and show that this sequence is Hox responsive. Our results support a mechanism in which Hox genes act upstream of Tbx5 to control the axial position of forelimb formation.

The forelimbs and hindlimbs of vertebrates are morphologically distinct. Pitx1, expressed in the hindlimb bud mesenchyme, is required for the formation of hindlimb characteristics and produces hindlimb-like morphologies when misexpressed in forelimbs. Pitx1 is also necessary for normal expression of Tbx4, a transcription factor required for normal hindlimb development. Despite the importance of this protein in these processes, little is known about its mechanism of action. Using a transgenic gene replacement strategy in a Pitx1 mutant mouse, we have uncoupled two discrete functions of Pitx1. We show that, firstly, this protein influences hindlimb outgrowth by regulating Tbx4 expression levels and that, subsequently, it shapes hindlimb bone and soft tissue morphology independently of Tbx4. We provide the first description of how Pitx1 sculpts the forming hindlimb skeleton by localised modulation of the growth rate of discrete elements.

While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous, as the limb progenitors differentiate, each individual tissue element attains a characteristic limb‐type morphology that ultimately defines the constitution of the forelimb or hindlimb. This review focuses on contemporary understanding of the regulation of limb bud initiation and formation of limb‐type specific morphologies and how these regulatory mechanisms evolved in vertebrates. We also attempt to clarify the definition of the terms limb‐type identity and limb‐type morphology that have frequently been used interchangeably. Over the last decade, three genes, Tbx4, Tbx5, and Pitx1, have been extensively studied for their roles in limb initiation and determining limb‐type morphologies. The role of Tbx4 and Tbx5 in limb initiation is clearly established. However, their putative role in the generation of limb‐type morphologies remains controversial. In contrast, all evidence supports a function for Pitx1 in determination of hindlimb morphologies. Developmental Dynamics 240:1017–1027, 2011.

Paired fins/limbs are one of the most successful vertebrate innovations, since they are used for numerous fundamental activities, including locomotion, feeding, and breeding. Gene duplication events generate new genes with the potential to acquire novel functions, and two rounds of genome duplication took place during vertebrate evolution. The cephalochordate amphioxus diverged from other chordates before these events and is widely used to deduce the functions of ancestral genes, present in single copy in amphioxus, compared to the functions of their duplicated vertebrate orthologues. The T-box genes Tbx5 and Tbx4 encode two closely related transcription factors that are the earliest factors required to initiate forelimb and hind limb outgrowth, respectively. Since the genetic components proposed to be responsible for acquiring a trait during evolution are likely to be involved in the formation of that same trait in living organisms, we investigated whether the duplication of an ancestral, single Tbx4/5 gene to give rise to distinct Tbx4 and Tbx5 genes has been instrumental in the acquisition of limbs during vertebrate evolution. We analyzed whether the amphioxus Tbx4/5 gene is able to initiate limb outgrowth, and assayed the amphioxus locus for the presence of limb-forming regulatory regions. We show that AmphiTbx4/5 is able to initiate limb outgrowth and, in contrast, that the genomic locus lacks the regulatory modules required for expression that would result in limb formation. We propose that changes at the level of Tbx5 and Tbx4 expression, rather than the generation of novel protein function, have been necessary for the acquisition of paired appendages during vertebrate evolution.

Background: The developing mouse limb is widely used as a model system for studying tissue patterning. Despite this, few references are available that can be used for the correct identification of developing limb structures, such as muscles and tendons. Existing textual references consist of two-dimensional (2D) illustrations of the adult rat or mouse limb that can be difficult to apply when attempting to describe the complex three-dimensional (3D) relationship between tissues. Results: To improve the resources available in the mouse model, we have generated a free, web-based, interactive reference of limb muscle, tendon, and skeletal structures at embryonic day (E) 14.5 http://www.nimr.mrc.ac.uk/3dlimb/. The Atlas was generated using mouse forelimb and hindlimb specimens stained using immunohistochemistry to detect muscle and tendon. Limbs were scanned using Optical Projection Tomography (OPT), reconstructed to make 3D models and annotated using computer-assisted segmentation tools in Amira 3D Visualisation software. The annotated dataset is visualised using Java, JAtlasView software. Users click on the names of structures and view their shape, position and relationship with other structures within the 3D model and also in 2D virtual sections. Conclusion: The Mouse Limb Anatomy Atlas provides a novel and valuable tool for researchers studying limb development and can be applied to a range of research areas, including the identification of abnormal limb patterning in transgenic lines and studies of models of congenital limb abnormalities. By using the Atlas for "virtual" dissection, this resource offers an alternative to animal dissection. The techniques we have developed and employed are also applicable to many other model systems and anatomical structures.

The Notch family of receptors is involved in a wide variety of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during cell differentiation and tissue morphogenesis. Notch1 and Notch ligands are expressed in the developing limbs, and Notch signalling has been implicated in the formation of a variety of tissues that comprise the limb, such as the skeleton, musculature, and vasculature. Notch signalling has also been implicated in regulating overall limb size. We have used a conditional allele of Notch1 in combination with two different Cre transgenic lines to delete Notch1 function either in the limb mesenchyme or in the apical ectodermal ridge (AER) and limb ectoderm. We demonstrate that Notch signalling, involving Notch1 and Jagged2, is required to regulate the number of Fgf8‐expressing cells that comprise the AER and that regulation of the levels of fibroblast growth factor signalling is important for the freeing of the digits during normal limb formation. Regulation of the extent of the AER is achieved by Notch signalling positively regulating apoptosis in the AER. We also demonstrate that Notch1 is not required for proper formation of all the derivatives of the limb mesenchyme. Developmental Dynamics 234:1006–1015, 2005.

Despite their obvious similarities, the forelimbs and hindlimbs of tetrapod vertebrates have evolved distinct structural elements to carry out their discrete functions. Many genes required for limb initiation and patterning are involved in regulatory networks common to both limb-types. Other genes are differentially expressed between forelimb and hindlimb, and have been implicated in the initiation of limb bud outgrowth and the specification of limb-type identity. In this review, I will discuss the current understanding of how genes that control limb identity interact with regulatory networks common to both appendages to produce the fingers of the hand and toes of the foot.

T-box genes are defined by the presence of a conserved sequence, the so-called T-box; this codes for the T-domain, which is involved in DNA-binding and protein dimerisation. Members of this gene family have been found in all metazoans, from diploblasts to humans, and mutations in T-box gene family members in humans have been linked to several congenital disorders. Sequencing of the complete genomes of a range of invertebrate and vertebrate species has allowed the classification of individual T-box genes into five subfamilies: Brachyury, T-brain1, Tbx1, Tbx2 and Tbx6. This review will largely focus on T-box genes identified in organisms whose genomes have been fully sequenced, emphasising how comparative studies of the T-box gene family will help to reveal the roles of these genes during development and in the adult.