Induction and patterning of the mesodermal germ layer is a key early step of vertebrate embryogenesis. We report that FoxD3 function in the Xenopus gastrula is essential for dorsal mesodermal development and for Nodal expression in the Spemann organizer. In embryos and explants, FoxD3 induced mesodermal genes, convergent extension movements and differentiation of axial tissues. Engrailed-FoxD3, but not VP16-FoxD3, was identical to native FoxD3 in mesoderm-inducing activity, indicating that FoxD3 functions as a transcriptional repressor to induce mesoderm. Antagonism of FoxD3 with VP16-FoxD3 or morpholinoknockdown of FoxD3 protein resulted in a complete block to axis formation, a loss of mesodermal gene expression, and an absence of axial mesoderm, indicating that transcriptional repression by FoxD3 is required for mesodermal development. FoxD3 induced mesoderm in a non-cell-autonomous manner, indicating a role for secreted inducing factors in the response to FoxD3. Consistent with this mechanism, FoxD3 was necessary and sufficient for the expression of multiple Nodal-related genes, and inhibitors of Nodal signaling blocked mesoderm induction by FoxD3. Therefore, FoxD3 is required for Nodal expression in the Spemann organizer and this function is essential for dorsal mesoderm formation. KEY WORDS:Xenopus, FoxD3, Forkhead, Nodal, Mesoderm, Transcription Development 133, 4827-4838 (2006) DEVELOPMENT 4828 formation, and antagonism or knockdown of FoxD3 results in severe axial defects and loss of dorsal mesodermal gene expression. FoxD3 induction of mesoderm is non-cell-autonomous and requires the Nodal signaling pathway. Consistent with the co-expression of FoxD3 and Nodal genes in the organizer, FoxD3 is necessary and sufficient for the expression of several Nodal-related genes. Taken together, our results demonstrate a novel mode of Nodal regulation in the Spemann organizer, where transcriptional repression by FoxD3 maintains Nodal expression to promote mesoderm induction and axial development. MATERIALS AND METHODS Embryos and microinjectionEmbryos were collected, fertilized, injected and cultured as previously described (Yao and Kessler, 1999), and embryonic stage was determined according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Dorsal and ventral blastomeres were identified by pigmentation differences (Klein, 1987). Explants were prepared using a Gastromaster microsurgery instrument (Xenotek Engineering). Capped, in vitro transcribed RNA for microinjection was synthesized from linearized template DNA using the Message Machine kit (Ambion) and 10 nl of RNA solution was injected.Templates for in vitro transcription were pCS2-FoxD3, pCS2-mFoxD3, pCS2-Eng-FoxD3, pCS2-VP16-FoxD3, pCS2-FoxD3(N140A/H144A), pCS2-Eng-FoxD3(N140A/H144A), pCS2-VP16-FoxD3(N140A/H144A), pCS2-NLS-FoxD3WH, pCS2-FoxD3-utr (this study), pCS2-Eng, pCS2-VP16 (Kessler, 1997), pCS2-MT-SID (Chen et al., 1997), pCS2-Cer-S (Piccolo et al., 1999), pCS2-Xnr1 (Sampath et al., 1997), and pCS2-VegT⌬UTR (Engleka et al., 2001). FoxD3 expression ...

Causality assessment for suspected drug-induced liver injury (DILI) during drug development and following approval is challenging. The IQ DILI Causality Working Group (CWG), in collaboration with academic and regulatory subject matter experts (SMEs), developed this manuscript with the following objectives: (1) understand and describe current practices; (2) evaluate the utility of new tools/methods/practice guidelines; (3) propose a minimal data set needed to assess causality; (4) define best practices; and (5) promote a more structured and universal approach to DILI causality assessment for clinical development. To better understand current practices, the CWG performed a literature review, took a survey of member companies, and collaborated with SMEs. Areas of focus included best practices for causality assessment during clinical development, utility of adjudication committees, and proposals for potential new avenues to improve causality assessment. The survey and literature review provided renewed understanding of the complexity and challenges of DILI causality assessment as well as the use of non-standardized approaches. Potential areas identified for consistency and standardization included role and membership of adjudication committees, standardized minimum dataset, updated assessment tools, and best practices for liver biopsy and rechallenge in the setting of DILI. Adjudication committees comprised of SMEs (i.e., utilizing expert opinion) remain the standard for DILI causality assessment. A variety of working groups continue to make progress in pursuing new tools to assist with DILI causality assessment. The minimum dataset deemed adequate for causality assessment provides a path forward for standardization of data collection in the setting of DILI. Continued progress is necessary to optimize and advance innovative tools necessary for the scientific, pharmaceutical, and regulatory community. The views expressed are those of the authors and do not necessarily represent the position of, nor imply endorsement from, any of the companies, the US Food and Drug Administration or the US Government.

Abstractα2-macroglobulin is a major serum protein with diverse functions, including inhibition of protease activity and binding of growth factors, cytokines, and disease factors. We have cloned and characterized Panza, a new Xenopus laevis α2-macroglobulin. Panza has 56-60% amino acid similarity with previously identified Xenopus, mouse, rat and human α2-macroglobulins, indicating that Panza is a new member of the α2-macroglobulin family. Panza mRNA is first detected at the beginning of neurulation in the dorsal endoderm lining the primitive gut (archenteron roof). At the completion of neurulation and continuing through the late tadpole stage, Panza is restricted to a dorsal domain of the gut endoderm adjacent to the notochord and extending along the entire anteriorposterior axis. With outgrowth of the tailbud, Panza expression persists in the chordaneural hinge at the posterior end of the differentiating notochord and extends into the floor plate of the posterior neural tube. As gut coiling commences, Panza expression is initiated in the liver, and the dorsal domain of Panza expression becomes limited to the midgut and hindgut. With further gut coiling, strong Panza expression persists in the liver, but is lost from other regions of the gut. The expression of Panza in endodermal cells adjacent to the notochord points to a potential role for Panza in signal modulation and/or morphogenesis of the primitive gut. KeywordsXenopus laevis; α2-macroglobulin; endoderm; gut; digestive tract; liver Results and Discussionα2-macroglobulin (α 2 M) is an abundant plasma protein of vertebrates and arthropods that has a remarkable capacity to bind numerous and diverse ligands. α 2 M was first identified as a "panprotease inhibitor" capable of binding nearly all extracellular proteases, leading to clearance and degradation of α 2 M-protease complexes. Binding of protease to native α 2 M results in cleavage and activation of α 2 M, causing a conformational change that entraps protease and exposes binding sites for the α 2 M receptor. The α 2 M-protease complex binds to low density lipoprotein receptor-related protein (LRP), the major cell surface receptor for α 2 M, and the receptor bound complex is internalized by endocytosis, and targeted for lysosomal degradation (reviewed in Borth, 1992).* Author for correspondence Email:kesslerd@mail.med.upenn.edu, Tel: 215-898-1478, Fax: 215-573-7601 The conformational change that occurs with α 2 M activation also exposes binding sites for nonprotease ligands, including PDGF (Huang et al., 1984), FGF (Mathew et al., 2003), TGFβ1 (Huang et al., 1988;Stouffer et al., 1993;Feige et al., 1996;Arandjelovic et al., 2003), Activin A (Niemuller et al., 1995;Mather, 1996;Phillips, 2000), NGF (Ronne et al., 1979), TNF (James et al., 1992) and multiple Interleukins (Borth and Luger, 1989;Borth et al., 1990;Garber et al., 2000;Kurdowska et al., 2000). In complex with these ligands, α 2 M can serve either as a carrier that stabilizes ligand in circulation, a clearance factor for ligand degradation, or ...