In prior efforts, we contributed to a large body of work that described an essential role for Fibroblast Growth Factor (FGF) signaling during limb development by examining mice lacking genes that encode FGF ligands and receptors. Another important signaling pathway during limb development controlled by Bone Morphogenetic Proteins (BMPs), which arguably controls all aspects of limb outgrowth: early patterning in all three axes, programmed cell death and bone formation. Therefore we have set ourselves the task to understand how BMP and FGF signaling pathways interact during limb development.Because of our general interest in limb development we have participated in a collaboration that demonstrated that Islet1-mediated activation of the beta-catenin pathway is necessary for hindlimb initiation. In second collaboration it was determined that the Dicer gene product is required for the proper positioning of the hindlimb bud along the anterior -posterior body axis (Zhang, Z. et al 2011 Dev Biol 351, 254). Finally, also in a collaborative study, we determined that all of the elements of the pelvic girdle and its surrounding perichondrium are Tbx4-derived. Also, although Tbx4 is thought to be associated primarily with the hindlimb, we characterized two forelimb expression domains in a study in which we reexamined the expression of Tbx4 in detail and also traced the fates of Tbx4-expressing cells with a previously generated, but incompletely characterized, allele of Tbx4 knocked in Cre allele.In current work, we are studying the role of BMP signaling as effectors of normal programmed cell death that occurs in mesenchymal interdigit cells, thus removing them and sculpting the final digit pattern in animals that are born without webbed limbs. In previous work produced genetic evidence for a novel model in which the surface ectoderm must receive a BMP signal, resulting in down regulation of Fgfs which in turn induces apoptosis of the underlying mesenchyme. Thus we demonstrated that BMPs control programmed cell death indirectly, by regulating FGF signaling. However, it is important to emphasize that this insight does not exclude a direct role for BMP signaling in controlling cell death in the developing limb. Therefore we are extended these studies by studying the role of BMP and FGF signaling in various aspects of limb development using mouse lines that express Cre in specific region of the developing limb. For example the only way to test the hypothesis that BMPs act as direct effectors of cell death is to inactivate BMPs receptors only in the lineage that undergoes cells death, without affecting FGF expression in nearby cells. We have achieved this using new Cre lines that allow Cre-mediated gene inactivation in these lineages. With these lines are asking: are BMPs are direct effectors of normal programmed cell death? If not, how is programmed cell death controlled? If so, how do BMPs achieve this endpont?In a serendipitous discovery, we have found that removal of a BMP signal to the limb bud interdigit zone rescues the requirement for a BMP signal to the digit region of the developing limb. Our efforts to understand this rescue may lead to a fundamental understanding of patterning in the developing limb. In another study, we have uncovered an important node of signaling between FGFs and BMP that is essential for normal development of the limb skeleton. Our previous work, cited above, demonstrates that specific FGFs, secreted from a distal structure in the limb bud, regulate the normal outgrowth and patterning of the limb. In current work, we are generating genetic evidence that BMP signaling to the progenitor population of the skeletal elements regulates this FGF signal by controlling the expression of an FGF antagonist. This linking of the two signaling pathways is not only a unique insight into how the limb is patterned but may provide a model for how the two pathways interact in other developmental contexts or during cancer.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Kaltcheva, Maria M; Anderson, Matthew J; Harfe, Brian D et al. (2016) BMPs are direct triggers of interdigital programmed cell death. Dev Biol 411:266-76
Hung, Irene H; Schoenwolf, Gary C; Lewandoski, Mark et al. (2016) A combined series of Fgf9 and Fgf18 mutant alleles identifies unique and redundant roles in skeletal development. Dev Biol 411:72-84
Mackem, Susan; Lewandoski, Mark (2011) Development. Limb cells don't tell time. Science 332:1038-9
Kawakami, Yasuhiko; Marti, Merce; Kawakami, Hiroko et al. (2011) Islet1-mediated activation of the *-catenin pathway is necessary for hindlimb initiation in mice. Development 138:4465-73
Naiche, L A; Arora, Ripla; Kania, Artur et al. (2011) Identity and fate of Tbx4-expressing cells reveal developmental cell fate decisions in the allantois, limb, and external genitalia. Dev Dyn 240:2290-300
Zhang, Zhen; O'Rourke, Jason R; McManus, Michael T et al. (2011) The microRNA-processing enzyme Dicer is dispensable for somite segmentation but essential for limb bud positioning. Dev Biol 351:254-65
Lewandoski, Mark; Mackem, Susan (2011) Developmental biology: extending the limb and body with vectors and scalars. Curr Biol 21:R34-6
Mackem, Susan; Lewandoski, Mark (2009) Limb development takes a measured step toward systems analysis. Sci Signal 2:pe33
Lewandoski, Mark; Mackem, Susan (2009) Limb development: the rise and fall of retinoic acid. Curr Biol 19:R558-61
Tzchori, Itai; Day, Timothy F; Carolan, Peter J et al. (2009) LIM homeobox transcription factors integrate signaling events that control three-dimensional limb patterning and growth. Development 136:1375-85