1. Limb bud initiation and outgrowth in response to FGF and Wnt signals: 1A. Identification of T-box transcription factor Tbx5 targets during early limb bud outgrowth. The initiation of limb bud formation and induction of the apical ectodermal ridge (AER) that directs subsequent limb outgrowth appear to be regulated by a cascade of FGF and Wnt signaling molecules. Intermediate target genes in this signal relay are largely unknown, but mutant analyses have demonstrated that the related limb-type specific T-box transcription factors Tbx5 in forelimb, and Tbx4 in hindlimb are essential for initiating outgrowth and interact with both FGF and Wnt pathways. We are developing chromatin immunoprecipitation (ChIP) assays in embryos to enable direct identification of target promoters in vivo for T-box genes that regulate limb outgrowth. We have successfully piloted ChIP assays with embryo limb bud lysates using anti-Tbx5 and identified several new genes that are regulated by Tbx5 during limb bud outgrowth using a Nimblegen 1.5kb promoter array that includes over 20,000 mouse gene promoters. ChIP is now being scaled up using embryo limb bud lysates to interrogate a larger promoter array set covering more regulatory sequence upstream of each gene. Identification of additional Tbx5 targets in the early limb bud will provide new insights on key steps in initiation of limb outgrowth. A similar approach will be used for other transcription factors in the limb, for which we have also developed high-specificity polyclonal antibodies (Hoxd genes-see 3B below).1B. Role of canonical Wnt signals in limb initiation and regulation of limb dorso-ventral development. Canonical Wnt signaling is essential for induction of limb buds at particular sites along the body axis but Wnt signals can also use different (non-canonical) pathways. It has been proposed that the inductive response initiates in the limb mesoderm and later is transferred to the ectoderm during AER formation but recent genetic evidence suggests that only signaling to the ectoderm may be needed for induction. After the limb bud has formed, Wnt signals also regulate dorsal (vs ventral) fates of musculo-skeletal components of the limb (ie. back of hand vs palm surface); whether this occurs through the canonical pathway that activates beta-catenin, or a non-canonical pathway has remained controversial. In a collaborative project with Dr. Yingzi Yang (NHGRI) we are crossing tissue-specific inducible Cre recombinase lines that we have generated with a conditional (floxed) beta-catenin allele to determine the role of the canonical Wnt pathway in specific tissues during limb induction and dorso-ventral patterning. 2. Regulation of limb anterior-posterior (AP) patterning to produce distinct digit identities (morphologies) by Shh, Hoxd genes and late signaling:2A. Nature of Shh function during limb development. Sonic Hedgehog (Shh) is a signaling molecule that acts as a mitogen and cell survival factor in many adult processes and is also thought to act as a morphogen that determines different cell fates in the developing embryo, depending on dosage of Shh that different cells encounter. In the limb, Shh is thought to regulate both digit number and digit pattern, producing distinct identities of different digits from anterior to posterior (ie. thumb to pinky). Shh has long been thought to act as a morphogen forming a spatial gradient along the AP axis of the limb bud, with higher concentrations of signal specifying more posterior digit types. Since many of the posterior digits are descended from cells that previously expressed Shh before proliferating and moving out of the Shh-expressing zone, it has recently been proposed that Shh acts in a temporal rather than spatial gradient and digit precursors that are exposed to autocrine Shh signals for the longest time duration become the most posterior digits.
