EARLY CHICK LIMB CARTILAGINOUS ELEMENTS POSSESS POLARIZING ACTIVITY AND EXPRESS HEDGEHOG-RELATED MORPHOGENETIC FACTORS

Skeletal patterning and morphogenesis in the developing limb are thoug ht to be regulated by instructive factors and cues from the zone of po larizing activity (ZPA), the apical ectodermal ridge (AER), and the do rsal ectoderm. However, the activities of the ZPA and AER dwindle earl y in embryogenesis and soon after cease, when in fact the proximal ske letal elements are still rudimentary in structure and the more distal ones are yet to become recognizable. Thus, we asked whether the chondr ocytes emerging within each mesenchymal condensation may themselves st art expressing properties similar to those of ZPA and/or AER and, in s o doing, may bring skeletal development to completion. Indeed, we foun d that the cartilaginous, but not precartilaginous, tissues in early c hick limbs possess ZPA-like properties. They expressed an endogenous f actor related to Sonic hedgehog (Shh), most likely Indian hedgehog (Ih h), and when fragments were grafted to the anterior margin of host sta ge 16-20 chick wing buds, they induced supernumerary skeletal elements (polarizing activity). The acquisition of polarizing activity by the cartilaginous structures followed clear proximo-to-distal and posterio r-to-anterior routes. Thus, (I) stage 25 cartilaginous humerus had pol arizing activity while stage 25 prospective radius did not, (2) poster iorly-located stage 29 ulna had stronger activity than anteriorly-loca ted stage 29 radius, and (3) ulna's diaphysis had stronger activity at stage 29 than 31 while radius's diaphysis was stronger at stage 31 th an 29. Prior to inducing extra digit formation, the cartilaginous graf ts induced Hoxd-12 and Hoxd-13 gene expression in adjacent competent m esenchymal tissue. Strikingly, the cartilaginous grafts induced also e xpression of Shh and polarizing activity in adjacent mesenchyme, which ZPA grafts cannot do; thus, the cartilaginous structures displayed ac tivities ''upstream'' of those of the ZPA. The results support our hyp othesis that chondrocytes may themselves direct skeletal morphogenesis . In so doing and as a result of their inductive activities, the cells may also have an important role in the completion of limb patterning and morphogenesis. (C) 1996 Wiley-Liss, Inc.