This a third revision of an application to test the hypothesis that two genes, midkine (Mdk) and heparin-binding neurotrophic factor (Hbnf), participate in the signaling cascades that orchestrate limb growth and patterning. Vogt initially chose to study these genes because their encoded protein products appear to meet two of three criteria expected of factors that mediate morphogenetic signals during limb development: responsiveness to retinoids and functional features of an extracellular signalling molecule. Experiments performed by the Vogt lab and described in Preliminary Studies indicate that Mdk and Hbnf also meet a third criteria, spatially restricted expression in regions of the limb bud known to have morphogenetic activity. Mdk was originally cloned in a screen for retinoic acid induced mRNAs in a mouse embryonal carcinoma cell line. Hbnf, which shares 50% amino acid homology with Mdk, was isolated in a search for mammalian heparin-binding proteins. Vogt proposes four parallel sets of experiments to determine whether Mdk and/or Hbnf play a role in limb morphogenesis. Published genetic mapping studies from Vogt's lab placed Mdk on mouse chromosome 2 near two developmental mutations, Ulnaless (Ul) and First Arch (Far); these same studies placed Hbnf on mouse chromosome 6 near the Sightless (Sig) and hop-sterile (hop) mutations. Recent studies discussed in Preliminary Studies have also shown that Strong's Luxoid (lst) is closely linked to Mdk. The first specific aim is to refine the map positions of Mdk and Hbnf and to test for allelism with these closely linked mutations. In the second specific aim, germ line null mutations will be constructed for Mdk and Hbnf by gene targeting in ES cells.
Specific aim 3 proposes to correlate patterns of expression of Mdk and Hbnf between 6.5-11.5 d.p.c. with the expression of a number of genes known to be transcribed in morphogenetically active regions of the primitive streak embryo, in the neural tube, and in the limb. Expression will be examined by in situ hybridization to whole mounts and histological sections and by immunohistochemistry. In addition, Vogt plans to examine Mdk and Hbnf expression in, respectively, the Hbnf and Mdk knockouts, and in limb deformity and Strong's luxoid, two limb mutants with apical ectodermal ridge defects.
In specific aim four, Vogt proposes to establish chick limb bud cultures in his lab to study Mdk and Hbnf function and their relationship to other morphogenetic signals. First, in situ hybridizations will be performed with chick Mdk and Hbnf to compare their patterns of expression in the chick to those in the mouse. Second, the effect on limb growth and patterning of alterations in the levels and sites of Mdk and Hbnf expression will be examined by delivering purified protein via heparin agarose beads to different regions of the developing chick limb bud. Third, Mdk and Hbnf expression will be monitored following ridge removal and addition of sources of polarizing activity, including posterior limb mesoderm, retinoic acid impregnated beads, and sonic hedgehog.
