Jan L. Christian

Identification of Smad7, a TGFβ-inducible antagonist of TGF-β signalling

TGF-β signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-β receptor induces phosphorylation of two such proteins, Smad2 and Smad3 (refs 2, 3, , 5, 6), which form hetero-oligomeric complex(es) with Smad4/DPC4 (refs 5, 6, 7, 8, 9, 10) that translocate to the nucleus,,,, where they then regulate transcriptional responses,. However, the mechanisms by which the intracellular signals of TGF-β are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6 (ref. 13). Transfection of Smad7 blocks responses mediated by TGF-β in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-β signalling. Smad7 associates stably with the TGF-β receptor complex, but is not phosphorylated upon TGF-β stimulation. TGFβ-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-β rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-β responses.

Injected Wnt RNA induces a complete body axis in Xenopus embryos

Studies in Xenopus have shown that growth factors of the TGF beta and Wnt oncogene families can mimic aspects of dorsal axis formation. Here we directly compare the inductive properties of two Wnt proteins by injecting synthetic mRNA into developing embryos. The results show that Wnt-1 and Xwnt-8 can induce a new and complete dorsal axis and can rescue the development of axis-deficient, UV-irradiated embryos. In contrast, activin mRNA injection induces only a partial dorsal axis that lacks anterior structures. These studies demonstrate that the mechanism of Wnt-induced axis duplication results from the creation of an independent Spemann organizer. The relationship between the properties of the endogenous dorsal inducer and the effects of Wnts and activins is discussed.

Daughters against dpp modulates dpp organizing activity in Drosophila wing development

The family of TGF-β signalling molecules play inductive roles in various developmental contexts. One member of this family, Drosophila Decapentaplegic (Dpp) serves as a morphogen that patterns both the embryo, and adult,. We have now isolated a gene, Daughters against dpp ( Dad), whose transcription is induced by Dpp. Dad shares weak homology with Drosophila Mad (Mothers against dpp), a protein required for transduction of Dpp signals. In contrast to Mad or the activated Dpp receptor, whose overexpression hyperactivates the Dpp signalling pathway, overexpression of Dad blocks Dpp activity. Expression of Dad together with either Mad or the activated receptor rescues phenotypic defects induced by each protein alone. Dad can also antagonize the activity of a vertebrate homologue of Dpp, bone morphogenetic protein (BMP-4; ref. 7), as evidenced by induction of dorsal or neural fate following overexpression in Xenopus embryos. We conclude that the pattern-organizing mechanism governed by Dpp involves a negative-feedback circuit in which Dpp induces expression of its own antagonist, Dad. This feedback loop appears to be conserved in vertebrate development.

Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.

Wnts are secreted signaling factors which influence cell fate and cell behavior in developing embryos. Overexpression in Xenopus laevis embryos of a Xenopus Wnt, Xwnt-8, leads to a duplication of the embryonic axis. In embryos ventralized by UV irradiation, Xwnt-8 restores expression of the putative transcription factor goosecoid, and rescues normal axis formation. In contrast, overexpression of Xwnt-5A in normal embryos generates defects in dorsoanterior structures, without inducing goosecoid or a secondary axis. To determine whether Xwnt-4 and Xwnt-11 fall into one of these two previously described classes of activity, synthetic mRNAs were introduced into animal caps, normal embryos, and UV-treated embryos. The results indicate that Xwnt-4, Xwnt-5A, and Xwnt-11 are members of a single functional class with activities that are indistinguishable in these assays. To investigate whether distinct regions of Xwnt-8 and Xwnt-5A were sufficient for eliciting the observed effects of overexpression, we generated a series of chimeric Xwnts. RNAs encoding the chimeras were injected into normal and UV-irradiated Xenopus embryos. Analysis of the embryonic phenotypes and goosecoid levels reveals that chimeras composed of carboxy-terminal regions of Xwnt-8 and amino-terminal regions of Xwnt-5A are indistinguishable from the activities of native Xwnt-8 and that are the reciprocal chimeras elicit effects indistinguishable from overexpression of native Xwnt-5A. We conclude that the carboxy-terminal halves of these Xwnts are candidate domains for specifying responses to Xwnt signals.

BMP-4 is proteolytically activated by furin and/or PC6 during vertebrate embryonic development

Bone morphogenetic protein‐4 (BMP‐4) is a multifunctional developmental regulator. BMP‐4 is synthesized as an inactive precursor that is proteolytically activated by cleavage following the amino acid motif ‐Arg‐Ser‐Lys‐Arg‐. Very little is known about processing and secretion of BMPs. The proprotein convertases (PCs) are a family of seven structurally related serine endoproteases, at least one of which, furin, cleaves after the amino acid motif ‐Arg‐X‐Arg/Lys‐Arg‐. To examine potential roles of PCs during embryonic development we have misexpressed a potent protein inhibitor of furin, α1‐antitrypsin Portland (α1‐PDX) in early Xenopus embryos. Ectopic expression of α1‐PDX phenocopies the effect of blocking endogenous BMP activity, leading to dorsalization of mesoderm and direct neural induction. α1‐PDX‐mediated neural induction can be reversed by co‐expression of downstream components of the BMP‐4 signaling pathway. Thus, α1‐PDX can block BMP activity upstream of receptor binding, suggesting that it inhibits an endogenous BMP‐4 convertase(s). Consistent with this hypothesis, α1‐PDX prevents cleavage of BMP‐4 in an oocyte translation assay. Using an in vitro digestion assay, we demonstrate that four members of the PC family have the ability to cleave BMP‐4, but of these, only furin and PC6B are sensitive to α1‐PDX. These studies provide the first in vivo evidence that furin and/or PC6 proteolytically activate BMP‐4 during vertebrate embryogenesis.

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF‐beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt‐8 signaling pathways in the induction and dorsal‐ventral patterning of mesoderm. Injection of Xwnt‐8 mRNA into 2‐cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non‐injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt‐8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV‐ventralized animals. Our results support a model whereby dorso‐ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal‐ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.

Effect of wnt-1 and related proteins on gap junctional communication in Xenopus embryos

The proto-oncogene wnt-1 (previously referred to as int-1) is thought to be important in embryonic pattern formation although its mechanisms of action are unknown. Premature and increased expression of the Wnt-1 protein, achieved by injection of synthetic wnt-1 RNA into fertilized Xenopus eggs, enhanced gap junctional communication between ventral cells of the developing embryo. This result is consistent with the hypothesis that Wnt proteins activate a receptor-mediated signal transduction pathway and that gap junctional communication can be a target of this pathway. The effects of two Wnt-1-related proteins on gap junctional communication were also investigated: overexpression of Xwnt-8 increased gap junctional coupling in a manner similar to Wnt-1, whereas Xwnt-5A did not. These findings are consistent with the existence of multiple receptors for Wnt proteins.

Bone morphogenetic protein function is required for terminal differentiation of the heart but not for early expression of cardiac marker genes

To examine potential roles for bone morphogenetic proteins (BMPs) in cardiogenesis, we used intracellular BMP inhibitors to disrupt this signaling cascade in Xenopus embryos. BMP-deficient embryos showed endodermal defects, a reduction in cardiac muscle-specific gene expression, a decrease in the number of cardiomyocytes and cardia bifida. Early expression of markers of endodermal and precardiac fate, however, was not perturbed. Heart defects were observed even when BMP signal transduction was blocked only in cells that contribute primarily to endodermal, and not cardiac fates, suggesting a non-cell autonomous function. Our results suggest that BMPs are not required for expression of early transcriptional regulators of cardiac fate but are essential for migration and/or fusion of the heart primordia and cardiomyocyte differentiation.

Fanconi Anemia Proteins Are Required To Prevent Accumulation of Replication-Associated DNA Double-Strand Breaks

ABSTRACT Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.

Smad6 functions as an intracellular antagonist of some TGF‐β family members during Xenopus embryogenesis

Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signalling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation‐mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription.