Faculty Bibliography

The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5-E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral border nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb

The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. We have investigated the in vivo role that such acylation might play in appendage patterning in mouse and Drosophila; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species.

Patterning of the limb is coordinated by the complex interplay of three signaling regions: the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the non-ridge limb ectoderm. Complex feedback loops exist between Shh in the ZPA, Bmps and their antagonists in the adjacent mesenchyme, Wnt7a in the dorsal ectoderm and Fgfs in the AER. In contrast to the previously reported complete absence of digits in Shh(-/-) mice, we show that one morphologically distinct digit, with a well-delineated nail and phalanges, forms in Shh(-/-) hindlimbs, while intermediate structures are severely truncated and fused. The presence of distal autopod elements is consistent with weak expression of Hoxd13 in Shh(-/-) hindlimbs. Shh(-/-) forelimbs in contrast have one distal cartilage element, a less-well differentiated nail and fused intermediate bones. Interestingly, Ihh is expressed at the tip of Shh mutant limbs and could account for formation of distal structures. In contrast to previous studies we also demonstrate that Shh signaling is required for maintenance of normal Fgf8 expression, since expression of Fgf8, unlike some other AER marker genes, is rapidly lost from anterior to posterior after E10.5, with only a small domain of Fgf8 expression remaining posteriorly. Furthermore, loss of expanded Fgf8 expression is paralleled by a collapse of the handplate. Our data show that development of most intermediate elements of the hindlimb skeleton are Shh-dependent, and that Shh signaling is required for anterior-posterior expansion of the AER in both limbs and for the subsequent branching of zeugopod and autopod elements. Finally, we show that Shh is also required for outgrowth of the limb ectoderm and thus for the formation of a distinct limb compartment

Rat skin was exposed to the plateau region of the 1.0 GeV/nucleon 56Fe beam at the Brookhaven AGS. Rats were irradiated or not with single of split doses of 56Fe or argon; some 56Fe-exposed rats were fed 250 ppm retinyl acetate continuously in the lab chow beginning 1 week before irradiation. All lesions were noted, photographed and identified for eventual histological diagnosis. The preponderance of the tumors so far are fibromas. The data show that single doses of 56Fe ions are 2 or 3 fold more effective than argon in producing tumors at 4.5 Gy but are about equally effective at 3.0 Gy and 9.0 Gy. The presence of 250 ppm retinyl acetate in the lab chow reduced the incidence of tumors by about 50-60% in comparison to groups exposed only to the radiation. These are preliminary findings based on only about one-fourth the eventual number of tumors expected