Characterization of zebrafish smad1, smad2 and smad5: the amino-terminus of smad1 and smad5 is required for specific function in the embryo.

Members of the TGFbeta superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGFbeta signalling, smad1, 2 and 5, from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGFbeta signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGFbeta signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGFbeta ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGFbeta members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.     

Role of the anterior visceral endoderm in restricting posterior signals in the mouse embryo

Recent genetic and embryological experiments have demonstrated that head formation in the mouse embryo is dependent on signals provided by two organising centers during gastrulation, the anterior visceral endoderm (AVE) and the anterior primitive streak (also called the Early Gastrula Organiser, EGO). However the molecular nature of the signals triggering anterior neural formation from the epiblast is not clearly understood. The analysis of mouse mutants has allowed the identification of some of the molecular players involved in the process of head formation. In this review, we describe different mutant embryos in which impairment of visceral endoderm function leads to similar defects in antero-posterior axis specification. These phenotypes are consistent with a role of the AVE in protecting anterior embryonic regions from signals that promote posterior development. We propose that a genetic cascade in the AVE, involving HNF3beta, Lim1, Otx2, Smad2 and ActRIB, leads to the production of secreted TGFbeta antagonists that protect the anterior epiblast region from Nodal signalling.     

Smad signalling in the ovary

It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context.     

Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo

Genetic and biochemical data have identified Smad4 as a key intracellular effector of the transforming growth factor beta (TGFbeta superfamily of secreted ligands. In mouse, Smad4-null embryos do not gastrulate, a phenotype consistent with loss of other TGFbeta-related signaling components. Chimeric analysis reveals a primary requirement for Smad4 in the extra-embryonic lineages; however, within the embryo proper, characterization of the specific roles of Smad4 during gastrulation and lineage specification remains limited. We have employed a Smad4 conditional allele to specifically inactivate the Smad4 gene in the early mouse epiblast. Loss of Smad4 in this tissue results in a profound failure to pattern derivatives of the anterior primitive streak, such as prechordal plate, node, notochord and definitive endoderm. In contrast to these focal defects, many well-characterized TGFbeta- and Bmp-regulated processes involved in mesoderm formation and patterning are surprisingly unaffected. Mutant embryos form abundant extra-embryonic mesoderm, including allantois, a rudimentary heart and middle primitive streak derivatives such as somites and lateral plate mesoderm. Thus, loss of Smad4 in the epiblast results not in global developmental abnormalities but instead in restricted patterning defects. These results suggest that Smad4 potentiates a subset of TGFbeta-related signals during early embryonic development, but is dispensable for others.     

SMA-3 smad has specific and critical functions in DBL-1/SMA-6 TGFbeta-related signaling

A TGFbeta signal transduction cascade controls body size and male tail morphogenesis in the nematode Caenorhabditis elegans. We have analyzed the function of the sma-3 Smad gene, one of three Smad genes that function in this pathway. Null mutations in sma-3 are at least as severe as null mutations in the ligand and type I receptor genes, dbl-1 and sma-6, indicating that the other Smads do not function in the absence of SMA-3. Furthermore, null mutations in sma-3 do not cause defects in egg laying or in regulation of the developmentally arrested dauer larva stage, indicating no overlapping function with another C. elegans TGFbeta signaling pathway. The sma-3 gene is widely expressed at all developmental stages in hermaphrodites and males. The molecular lesions associated with eight sma-3 alleles of varying severity have been determined. The missense mutations cluster in two previously identified regions important for Smad function.     

The interaction of endoglin with beta-arrestin2 regulates transforming growth factor-beta-mediated ERK activation and migration in endothelial cells

In endothelial cells, transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation and migration, the ALK-1/Smads 1/5/8 pathway and the ALK-5/Smads 2/3 pathway. TGF-beta signaling through these pathways is further regulated in endothelial cells by the endothelial specific TGF-beta superfamily co-receptor, endoglin. The importance of endoglin, ALK-1, and ALK-5 in endothelial biology is underscored by the embryonic lethal phenotypes of knock-outs in mice due to defects in angiogenesis, and by the presence of disease-causing mutations in these genes in human vascular diseases. However, the mechanism of action of endoglin is not well defined. Here we define a novel interaction between endoglin and the scaffolding protein beta-arrestin2. Both co-immunoprecipitation and fluorescence confocal studies demonstrate the specific interaction between endoglin and beta-arrestin2 in endothelial cells, enhanced by ALK-1 and to a lesser extent by the type II TGF-beta receptor. The endoglin/beta-arrestin2 interaction results in endoglin internalization and co-accumulation of endoglin and beta-arrestin2 in endocytic vesicles. Whereas endoglin did not have a direct impact on either Smad 2/3 or Smad 1/5/8 activation, endoglin antagonized TGF-beta-mediated ERK signaling, altered the subcellular distribution of activated ERK, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with beta-arrestin2. Reciprocally, small interfering RNA-mediated silencing of endogenous beta-arrestin2 expression restored TGF-beta-mediated ERK activation and increased endothelial cell migration in an endoglin-dependent manner. These studies define a novel function for endoglin, and further expand the roles mediated by the ubiquitous scaffolding protein beta-arrestin2.     

Mouse smad8 phosphorylation downstream of BMP receptors ALK-2, ALK-3, and ALK-6 induces its association with Smad4 and transcriptional activity

Smads are intracellular signaling mediators for TGF-beta superfamily. Smad1 and Smad5 are activated by BMP receptors. Here, we have cloned mouse Smad8 and functionally characterized its ability to transduce signals from BMP receptors. Constitutively active BMP type I receptors, ALK-3 and ALK-6, as well as ALK-2, were phosphorylated Smad8 and induced Smad8 interaction with Smad4. Nuclear translocation of Smad8 was stimulated by constitutively active BMP type I receptors. In contrast, constitutively active TGF-beta type I receptor, ALK-5, did not exhibit any action on Smad8. Smad8 and Smad4 cooperatively induced the promoter of Xvent2, a homeobox gene that responds specifically to BMP signaling. Dominant-negative Smad8 was shown to inhibit the increase of alkaline phosphatase activity induced by BMP-2 on pluripotent mesenchymal C3H10T1/2 and myoblastic C2C12 cell lines. The presence of Smad8 mRNA in mouse calvaria cells and osteoblasts suggests a role of Smad8 in the osteoblast differentiation and maturation.     

Dynamic regulation of Smad expression during mesenchyme to epithelium transition in the metanephric kidney

Genetic studies have revealed a role for the transforming growth factor beta (TGFbeta) superfamily growth factors in development of the mouse kidney. In this study, we have characterized developmental expression of Smad proteins, the downstream effectors of TGFbeta superfamily ligands. Immunohistochemistry detecting Smads 1-5 and 8 was performed on 11.25-15.5 dpc kidney sections. We find that Smad proteins are readily detected in metanephric mesenchyme but are coordinately down-regulated in condensing mesenchyme and derivative structures such as renal vesicles. In order to define the stage at which down-regulation occurs, we used the heterologous inducer lithium chloride to promote condensation in isolated cultured mesenchymal explants and subsequently assayed expression of Smad 4. Down-regulation is complete in lithium chloride induced mesenchymal condensates, indicating that Smad regulation takes place at an early stage, prior to mesenchyme to epithelium transition.     

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination

During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade.