The chickoligozeugodactyly(ozd) mutant lacks sonic hedgehog function in the limb

Abstract

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.

Conditional inactivation of Fgfr1 in mouse defines its role in limb bud establishment, outgrowth and digit patterning

“…A plausible explanation for this change is that early phase and late phase Hoxd13 expression may be differentially controlled. This was also exemplified by a previous observation that in a limb-specific Shh chick mutant termed oligozeugodactly (ozd), early phase Hoxd13 expression is absent while late phase expression is present (Ros et al, 2003). In summary, our molecular data suggest that a combination of these gene expression changes may account for the phenotypes in the Tcre;Fgfr1 mutant.…”

Anteroposterior patterning in the limb and digit specification: Contribution of mouse genetics

“…Accordingly, limbs (and fins in the zebrafish) manifest a prepattern along the AP axis, in the absence of Shh (Zuniga et al, 1999a;Chiang et al, 2001;Kraus et al, 2001;Ros et al, 2003). Several pathways have been invoked to explain this prepattern and the subsequent correct localization of Shh, which are reviewed below.…”

“…4) are initially expressed over the whole limb bud, whereas Hoxd10-Hoxd13 are expressed in a posteriorly restricted domain from the onset of their expression (Nelson et al, 1996;Zakany et al, 2004). This early pattern is Shh-independent (Grieshammer et al, 1996;Ros et al, 1996;Neumann et al, 1999;Kraus et al, 2001;Ros et al, 2003). By inducing an inversion of the whole HoxD cluster, Zakany et al (2004) observed an ectopic expression of Hoxd11 and Hoxd13 over the entire limb bud, including the anterior domain, at early stages.…”

The chicken polydactyly (Po) locus causes allelic imbalance and ectopic expression of Shh during limb development

“…The action of the polarising region was first discovered using chick-grafting experiments that showed the polarising region could induce an extra set of digits with reversed antero-posterior polarity when grafted to the anterior margin of a host limb bud (Saunders and Gasseling, 1968). Application of Shh protein to the anterior of the developing chick limb also induces ectopic digits that are patterned in a concentrationand time-dependent manner (Yang et al, 1997) and absence of Shh expression in mice and chickens leads to loss of digits (Chiang et al, 1996;Ros et al, 2003). Therefore, normal digit number and pattern depend upon precise spatio-temporal regulation of Shh expression, which is known to be regulated by cell proliferation and cell death (Towers et al, 2008;Sanz-Ezquerro and Tickle, 2000) as well as mechanisms that modify the action of the Shh ligand (Lewis et al, 2001).…”

“…S1, which is available online), and a related upstream sequence that is associated with polydactyly in dogs has been termed the ''pZRS'' (Park et al, 2008). Meanwhile, the chicken OZD mutant, which lacks Shh expression in the limbs and has a loss of digits, is associated with intron3 of the LMBR1 gene (Ros et al, 2003;Maas and Fallon, 2004) as is human Acheiropodia in which a deletion between intron 3 and 4 of LMBR1 causes a loss of limb structures distal to the elbows and knees . Genetic evidence from mouse has shown that the ZRS sequence acts in cis with Shh (Lettice et al, 2002), physically interacting with the Shh sequence specifically in limb bud tissue to regulate Shh expression (Amano et al, 2009).…”