Elizabeth R. Farrell

Negative Feedback Regulation of FGF Signaling Levels by Pyst1/MKP3 in Chick Embryos

BACKGROUND The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.

Molecular interactions between Tbx3 and Bmp4 and a model for dorsoventral positioning of mammary gland development

The formation of the dorsoventral (DV) boundary is central to establishing the body plan in embryonic development. Although there is some information about how limbs are positioned along the DV axis and how DV skin color pattern is determined, the way in which mammary glands are positioned is unknown. Here we focus on Bmp4 and Tbx3, a gene associated with ulnar-mammary syndrome, and compare their expression along the DV axis in relation to mammary gland initiation in mouse embryos. Tbx3 is expressed in the mammary gland-forming region with Tbx15, a gene involved in a DV coat color being expressed more dorsally and Bmp4 being expressed more ventrally. When Tbx3 was overexpressed, formation of mammary gland epithelium was extended along the DV axis. In contrast, overexpression of Bmp4 inhibited both Tbx3 and Tbx15 expression. In addition, when BMP signaling was inhibited by NOGGIN, Lef1 expression was lost. Thus, we propose that mutual interactions between Bmp4 and Tbx3 determine the presumptive DV boundary and formation of mammary glands in early mouse embryogenesis. 1,19-Dioctadecyl-3,3,39,39-tetramethyl indocarbocyanine perchloride labeling experiments showed that cells associated with mammary glands originate more dorsally and then move ventrally. This finding, together with previous findings, suggests that the same DV boundary may not only position limbs and determine coat color but also position mammary glands. Furthermore, Bmp signaling appears to be a fundamental feature of DV patterning.

Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos

Expression of the gene encoding the MKP‐3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP‐3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP‐3/Pyst1 co‐localizes with activated ERK, and expression of MKP‐3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP‐3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF‐induced ERK activity.

Dynamic expression of Lef/Tcf family members and β-catenin during chick gastrulation, neurulation, and early limb development

Members of the Lef/Tcf family of HMG‐box transcription factors mediate the response to Wnt as part of the canonical Wnt signaling cascade. Positive and negative cofactors, including β‐catenin, CtBP, and Smad3, regulate the activity of Lef/Tcf transcription complexes. Interaction of Lef/Tcfs with β‐catenin results in target gene activation or repression, depending on the context. Here, we report the cloning of a novel chick Tcf‐1 splice variant and of a partial cDNA for chick Tcf‐3. We describe their expression patterns during early development and have compared them with the expression profiles of Lef‐1 and β‐catenin. We found restricted patterns during gastrulation, neurulation, somitogenesis, and early limb development. β‐catenin and Lef/Tcf expression did not always coincide, indicating developmental contexts in which Lef/Tcf proteins may interact with other cofactors and conversely, the areas in which β‐catenin may interact with other coregulators, or be involved in regulating adhesive properties of cells. Developmental Dynamics 229:703–707, 2004.

Cloning and expression of CSAL2, a new member of the spalt gene family in chick.

In this study we describe cloning and expression of CSAL2, a second member of the spalt gene family in chick. All spalt proteins are characterized by the presence of multiple zinc-finger motifs, which are highly conserved. Mutations in HSAL1, a human spalt gene result in Townes-Brocks syndrome (TBS). We show here that CSAL2 is expressed in many of the tissues affected in TBS, including neural tissue, limb buds, mesonephros and cloaca.