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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.