Analysis of talpid and wild-type chicken embryos reveals roles for Hedgehog signalling in development of the limb bud vasculature

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.

Generation of mice with functional inactivation oftalpid3, a gene first identified in chicken

“…2B,arrow and arrowhead;see Fig. S2 in the supplementary material), similar to the vascular defects observed in talpid 3 chicken (Davey et al, 2007). Heterozygous C57BL/6 talpid3 -/+ mice were crossed with CD1 mice.…”

“…The earlier demise of talpid3 -/-mouse embryos might be due to vascularisation defects [as previously described in chicken talpid 3 mutants (Davey et al, 2007)] impacting more severely on embryos with a placenta. talpid3 -/-mouse embryos display randomised left/right asymmetry with respect to heart looping, consistent with the loss of nodal cilia (for a review, see Hirokawa et al, 2009).…”

TheTalpid3gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation

“…Stage HH24 chicken embryos treated as described previously (Davey et al, 2007). For SEM, samples washed in 100% acetone for 10 minutes before critical point drying, flushed five times to ensure excess acetone was removed, dried, then placed on carbon mounts, coated with Au/Pd and imaged using an ESEM at 15 kV, 10 mm WD in high vacuum mode.…”

“…The chicken talpid 3 mutant has a complex phenotype, including polydactylous limbs with many unpatterned digits, vascular defects, hypoteleorism, abnormal dorsoventral patterning of the neural tube, loss of endochondral bone formation and embryonic lethality (Davey et al, 2007;Davey et al, 2006;Ede and Kelly, 1964a;Ede and Kelly, 1964b). Development of all the regions affected in the talpid 3 mutant embryo requires Hedgehog (Hh) signalling and analysis of the developing mutant limb bud and neural tube has shown that it is the response to Hh signalling that is defective (Lewis et al, 1999).…”

The chicken as a model for embryonic development

“…Talpid 3 embryos have large polydactylous limbs, (limbs with many digits) which have no digit identity ( Fig. 3 A), holoproscencephaly (a loss of midline structures in the face and head), and die after only 3-7 days of embryonic development due to severe vascular abnormalities (Davey et al, 2007). The accessibility of the chicken mutant to surgical manipulations such as recombination experiments between talpid 3 and wild-type limb bud tissue has shed light on the mutant phenotype.…”