Negative Smad Expression and Regulation in the Developing Chick Limb

The inhibitory or negative Smads, Smad6 and Smad7, block TGFβ superfamily signals of both the BMP and TGFβ classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

BACKGROUND: EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. CONCLUSION: EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs

Bare Bones Pattern Formation: A Core Regulatory Network in Varying Geometries Reproduces Major Features of Vertebrate Limb Development and Evolution

BACKGROUND: Major unresolved questions regarding vertebrate limb development concern how the numbers of skeletal elements along the proximodistal (P-D) and anteroposterior (A-P) axes are determined and how the shape of a growing limb affects skeletal element formation. There is currently no generally accepted model for these patterning processes, but recent work on cartilage development (chondrogenesis) indicates that precartilage tissue self-organizes into nodular patterns by cell-molecular circuitry with local auto-activating and lateral inhibitory (LALI) properties. This process is played out in the developing limb in the context of a gradient of fibroblast growth factor (FGF) emanating from the apical ectodermal ridge (AER). RESULTS: We have simulated the behavior of the core chondrogenic mechanism of the developing limb in the presence of an FGF gradient using a novel computational environment that permits simulation of LALI systems in domains of varying shape and size. The model predicts the normal proximodistal pattern of skeletogenesis as well as distal truncations resulting from AER removal. Modifications of the model's parameters corresponding to plausible effects of Hox proteins and formins, and of the reshaping of the model limb, bud yielded simulated phenotypes resembling mutational and experimental variants of the limb. Hypothetical developmental scenarios reproduce skeletal morphologies with features of fossil limbs. CONCLUSIONS: The limb chondrogenic regulatory system operating in the presence of a gradient has an inherent, robust propensity to form limb-like skeletal structures. The bare bones framework can accommodate ancillary gene regulatory networks controlling limb bud shaping and establishment of Hox expression domains. This mechanism accounts for major features of the normal limb pattern and, under variant geometries and different parameter values, those of experimentally manipulated, genetically aberrant and evolutionary early forms, with no requirement for an independent system of positional information

Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning.

Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development