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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD030707-04
Application #
2673721
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1995-07-26
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
2000-06-30
Support Year
4
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Princeton University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
002484665
City
Princeton
State
NJ
Country
United States
Zip Code
08544
Moran, Jennifer L; Shifley, Emily T; Levorse, John M et al. (2009) Manic fringe is not required for embryonic development, and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain. Dev Dyn 238:1803-12
Cole, Susan E; Levorse, John M; Tilghman, Shirley M et al. (2002) Clock regulatory elements control cyclic expression of Lunatic fringe during somitogenesis. Dev Cell 3:75-84
Prince, V E; Holley, S A; Bally-Cuif, L et al. (2001) Zebrafish lunatic fringe demarcates segmental boundaries. Mech Dev 105:175-80
Amet, L E; Lauri, S E; Hienola, A et al. (2001) Enhanced hippocampal long-term potentiation in mice lacking heparin-binding growth-associated molecule. Mol Cell Neurosci 17:1014-24
Cole, S E; Mao, M S; Johnston, S H et al. (2001) Identification, expression analysis, and mapping of B3galt6, a putative galactosyl transferase gene with similarity to Drosophila brainiac. Mamm Genome 12:177-9
Hicks, C; Johnston, S H; diSibio, G et al. (2000) Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2. Nat Cell Biol 2:515-20
Peichel, C L; Kozak, C A; Luyten, F P et al. (1998) Evaluation of mouse Sfrp3/Frzb1 as a candidate for the lst, Ul, and Far mutants on chromosome 2. Mamm Genome 9:385-7
Peichel, C L; Prabhakaran, B; Vogt, T F (1997) The mouse Ulnaless mutation deregulates posterior HoxD gene expression and alters appendicular patterning. Development 124:3481-92
Peichel, C L; Abbott, C M; Vogt, T F (1996) Genetic and physical mapping of the mouse Ulnaless locus. Genetics 144:1757-67