Mesendoderm induction and reversal of left-right pattern by mouse Gdf1, a Vg1-related gene

TGFbeta signals play important roles in establishing the body axes and germ layers in the vertebrate embryo. Vg1 is a TGFbeta-related gene that, due to its maternal expression and vegetal localization in Xenopus, has received close examination as a potential regulator of development in Xenopus, zebrafish, and chick. However, a mammalian Vg1 ortholog has not been identified. To isolate mammalian Vg1 we screened a mouse expression library with a Vg1-specific monoclonal antibody and identified a single cross-reactive clone encoding mouse Gdf1. Gdf1 is expressed uniformly throughout the embryonic region at 5.5-6.5 days postcoitum and later in the node, midbrain, spinal cord, paraxial mesoderm, lateral plate mesoderm, and limb bud. When expressed in Xenopus embryos, native GDF1 is not processed, similar to Vg1. In contrast, a chimeric protein containing the prodomain of Xenopus BMP2 fused to the GDF1 mature domain is efficiently processed and signals via Smad2 to induce mesendoderm and axial duplication. Finally, right-sided expression of chimeric GDF1, but not native GDF1, reverses laterality and results in right-sided Xnr1 expression and reversal of intestinal and heart looping. Therefore, GDF1 can regulate left-right patterning, consistent with the Gdf1 loss-of-function analysis in the mouse (C. T. Rankin, T. Bunton, A. M. Lawler, and S. J. Lee, 2000, Nature Genet. 24, 262-265) and a proposed role for Vg1 in Xenopus. Our results establish that Gdf1 is posttranslationally regulated, that mature GDF1 activates a Smad2-dependent signaling pathway, and that mature GDF1 is sufficient to reverse the left-right axis. Moreover, these findings demonstrate that GDF1 and Vg1 are equivalent in biochemical and functional assays, suggesting that Gdf1 provides a Vg1-like function in the mammalian embryo.     

GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses

Within the TGF-β superfamily, there are approximately forty ligands divided into two major branches: the TGF-β/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression. In contrast, GDF3 inhibits BMP signaling upon over-expression of GDF3 mRNA, as recombinant protein, and regardless of its dose. We therefore further characterized the mechanism through which GDF3 protein acts as a specific BMP inhibitor and found that the BMP inhibitory activity of GDF3 resides redundantly in the unprocessed, predominant form and in the mature form of the protein. These results confirm and extend the activity that we described for GDF3 and illuminate the experimental basis for the different observations of others. We suggest that GDF3 is either a bi-functional TGF-β ligand, or, more likely, that it is a BMP inhibitor that can artificially activate Nodal signaling under non-physiological conditions.     

Growth differentiation factor 11 signaling controls retinoic acid activity for axial vertebral development

Mice deficient in growth differentiation factor 11 (GDF11) signaling display anterior transformation of axial vertebrae and truncation of caudal vertebrae. However, the in vivo molecular mechanisms by which GDF11 signaling regulates the development of the vertebral column have yet to be determined. We found that Gdf11 and Acvr2b mutants are sensitive to exogenous RA treatment on vertebral specification and caudal vertebral development. We show that diminished expression of Cyp26a1, a retinoic acid inactivating enzyme, and concomitant elevation of retinoic acid activity in the caudal region of Gdf11^−/− embryos may account for this phenomenon. Reduced expression or function of Cyp26a1 enhanced anterior transformation of axial vertebrae in wild-type and Acvr2b mutants. Furthermore, a pan retinoic acid receptor antagonist (AGN193109) could lessen the anterior transformation phenotype and rescue the tail truncation phenotype of Gdf11^−/− mice. Taken together, these results suggest that GDF11 signaling regulates development of caudal vertebrae and is involved in specification of axial vertebrae in part by maintaining Cyp26a1 expression, which represses retinoic acid activity in the caudal region of embryos during the somitogenesis stage.     

The conditioned medium from a stable human GDF3-expressing CHO cell line, induces the differentiation of PC12 cells

Members of the transforming growth factor-β (TGF-β) superfamily have significant roles in the regulation of a wide variety of physiological processes. In our present work, phylogenetic tree analysis showed that human GDF3 (Growth and differentiation factor 3) and human GDF1 formed a subgroup of closely related molecules. Through quantitative real-time PCR analysis in different human tissues, GDF1 and GDF3 expression level had a big difference in brain. GDF3 could activate downstream signaling through associating with ALK7 (Activin receptor-like kinase 7) in a Cripto-dependent fashion. A CHO cell line stably transfected with the encoding sequence of GDF3, named CHO-GDF3, was established. Western blotting analysis demonstrated that GDF3 protein could be secreted into the medium from CHO cells and immunofluorescence experiment showed that GDF3 was mainly distributed in cytoplasm of the stable cell line, the primary hippocampal neurons, and brain tissues. Furthermore, the conditioned medium from CHO-GDF3 could reduce PC12 cell growth and induce cell differentiation. All these findings bring new insights into the functional study of GDF3.     

The role and regulation of GDF11 in Smad2 activation during tailbud formation in the Xenopus embryo

A key role for phosphorylation of Smad2 by TGFβ superfamily ligands in the axial patterning of early embryos is well established. The regulation and role of Smad2 signaling in post-neurula embryonic patterning, however, is less well understood. While a variety of TGFβ superfamily ligands are implicated in various stages of anterior–posterior patterning, the ligand GDF11 has been shown to have a particular role in post-gastrula patterning in the mouse. Mouse GDF11 is specifically localized to the developing tail and is essential for normal posterior axial patterning. Mature GDF11 ligand is inhibited by its own prodomain, and extracellular proteolysis of this prodomain is thought to be necessary for GDF11 activity. The contribution of this proteolytic regulatory mechanism to Smad activation during embryogenesis in vivo, and to the development of posterior pattern, has not been characterized. We investigate here the role of Xenopus GDF11 in the activation of Smad2 during the development of tailbud-stage embryos, and the role of this activation in larval development. We also demonstrate that the activity of BMP-1/Tolloid-like proteases is necessary for the normal GDF11-dependent activation of Smad2 phosphorylation during post-gastrula development. These data demonstrate that GDF11 has a central role in the activation of Smad2 phosphorylation in tailbud stage Xenopus embryos, and provide the first evidence that BMP-1/Tolloid-mediated prodomain cleavage is important for activation of GDF11 in vivo.     

The role of Smad signaling in vascular and hematopoietic development revealed by studies using genetic mouse models

Smads are intracellular mediators of transforming growth factor β (TGF-β) superfamily signaling. In this review, we focus on the genetic mouse models for Smad pathways, which have provided functional evidence regarding the complex circuitry in angiogenesis and hematopoiesis during development. In the early stages of vascular development, TGF-β signaling is a contri buting factor in angiogenesis and vascular maturation. Whereas in the later embryogenesis, selected molecules of Smad pathways, such as TGF-β type II receptor (TbRII), ALK5, and Smad5, seem to be dispensable for vessel morphogenesis and integrity. TGF-β signaling is not required in the induction of hematopoietic precursors from mesoderm, but inhibits the subsequent expansion of committed hematopoietic precursors. By contrast, bone morphogenetic protein 4 (BMP4) has long been acknowledged pivotal in mesoderm induction and hematopoietic commitment during development. However, recent genetic evidence shows the BMP4-ALK3 axis is not crucial for the formation of hematopoietic cells from FLK1+ mesoderm. Because of the highly redundant mechanisms within the Smad pathways, the precise role of the Smad signaling involved in vascular and hematopoietic development remains nebulous. The generation of novel cell lineage restricted Cre transgenes would shed new light on the future relevant investigations.     

Camptothecin effectively treats obesity in mice through GDF15 induction

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15’s anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15’s specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight in rodents and nonhuman primates. This study reveals that the small molecule Camptothecin induces endogenous GDF15, suppressing food intake and reducing body weight in obese mice, suggesting a promising candidate for anti-obesity treatment.     

The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo

The formation of the anterior visceral endoderm (AVE) in the pre-gastrulation mouse embryo represents a crucial event in patterning of the anterior-posterior axis. Here, we show that the transforming growth factor beta (Tgfbeta) family member Gdf3 (growth-differentiation factor 3), a close relative of Xenopus Vg1, resembles the Tgfbeta ligand Nodal in both its signaling activity and its role in AVE formation in vivo. Thus, in cell culture, Gdf3 signaling requires the EGF-CFC co-receptor Cripto and can be inhibited by Lefty antagonists. In Xenopus embryos, Gdf3 misexpression results in secondary axis formation, and induces morphogenetic elongation and mesendoderm formation in animal caps. In mouse embryos, Gdf3 is expressed in the inner cell mass and epiblast, and null mutants frequently exhibit abnormal formation or positioning of the AVE. This phenotype correlates with defects in mesoderm and definitive endoderm formation, as well as abnormal Nodal expression levels. Our findings indicate that Gdf3 acts in a Nodal-like signaling pathway in pre-gastrulation development, and provide evidence for the functional conservation of Vg1 activity in mice.     

TGF-b Superfamily Cytokine MIC-1/GDF15 Is a Physiological Appetite and Body Weight Regulator

The TGF-b superfamily cytokine MIC-1/GDF15 circulates in all humans and when overproduced in cancer leads to anorexia/cachexia, by direct action on brain feeding centres. In these studies we have examined the role of physiologically relevant levels of MIC-1/GDF15 in the regulation of appetite, body weight and basal metabolic rate. MIC-1/GDF15 gene knockout mice (MIC-1^−/−) weighed more and had increased adiposity, which was associated with increased spontaneous food intake. Female MIC-1^−/− mice exhibited some additional alterations in reduced basal energy expenditure and physical activity, possibly owing to the associated decrease in total lean mass. Further, infusion of human recombinant MIC-1/GDF15 sufficient to raise serum levels in MIC-1^−/− mice to within the normal human range reduced body weight and food intake. Taken together, our findings suggest that MIC-1/GDF15 is involved in the physiological regulation of appetite and energy storage.     

Cluap1 localizes preferentially to the base and tip of cilia and is required for ciliogenesis in the mouse embryo

Qilin is one of several genes in zebrafish whose mutation results in cystic kidney. We have now studied the role of its mouse ortholog, Cluap1, in embryonic development by generating Cluap1 knockout (Cluap1^−/−) mice. Cluap1^−/− embryos died mid-gestation manifesting impairment of ciliogenesis in various regions including the node and neural tube. The basal body was found to be properly docked to the apical membrane of cells in the mutant, but the axoneme failed to grow. Cluap1 is a ciliary protein and is preferentially localized at the base and tip of cilia. Hedgehog signaling, as revealed with a Pacthed1-lacZ reporter gene, was lost in Cluap1^−/− embryos at embryonic day (E) 8.5 but was ectopically expanded at E9.0. The Cluap1 knockout embryos also failed to manifest left–right asymmetric expression of Nodal in the lateral plate, most likely as a result of the loss of Hedgehog signaling in node crown cells that in turn leads to pronounced down-regulation of Gdf1 expression in these cells. Crown cell-specific restoration of Cluap1 expression rescued Gdf1 expression in crown cells and left-sided Nodal expression in the lateral plate of mutant embryos. Our results suggest that Cluap1 contributes to ciliogenesis by regulating the intraflagellar transport (IFT) cycle at the base and tip of the cilium.     

Cripto/GRP78 modulation of the TGF-β pathway in development and oncogenesis

Cripto is a small, GPI-anchored signaling protein that regulates cellular survival, proliferation, differentiation and migration during normal developmental processes and tumorigenesis. Cripto functions as an obligatory co-receptor for the TGF-β ligands Nodal, GDF1 and GDF3 but attenuates signaling of others such as activin-A, activin-B and TGF-β1. Soluble, secreted forms of Cripto also activate Src, ras/raf/MAPK and PI3K/Akt pathways via a mechanism that remains largely obscure. This review describes the biological roles and signaling mechanisms of Cripto, highlighting our identification of the 78 kDa glucose regulated protein (GRP78) as a cell surface receptor/co-factor required for Cripto signaling via both TGF-β and Src/MAPK/PI3K pathways. We discuss emerging evidence indicating that Cripto/GRP78 signaling regulates normal somatic stem cells and their tumorigenic counterparts.     

GDF11 modulates NGN3+ islet progenitor cell number and promotes beta-cell differentiation in pancreas development

Identification of endogenous signals that regulate expansion and maturation of organ-specific progenitor cells is a major goal in studies of organ development. Here we provide evidence that growth differentiation factor 11 (GDF11), a member of the TGF-beta ligand family, governs the number and maturation of islet progenitor cells in mouse pancreas development. Gdf11 is expressed in embryonic pancreatic epithelium during formation of islet progenitor cells that express neurogenin 3. Mice deficient for Gdf11 harbor increased numbers of NGN3+ cells, revealing that GDF11 negatively regulates production of islet progenitor cells. Despite a marked expansion of these NGN3+ islet progenitors, mice lacking Gdf11 have reduced beta-cell numbers and evidence of arrested beta-cell development, indicating that GDF11 is also required for beta-cell maturation. Similar precursor and islet cell phenotypes are observed in mice deficient for SMAD2, an intracellular signaling factor activated by TGF-beta signals. Our data suggest that Gdf11 and Smad2 regulate islet cell differentiation in parallel to the Notch pathway, which previously has been shown to control development of NGN3+ cells. Thus, our studies reveal mechanisms by which GDF11 regulates the production and maturation of islet progenitor cells in pancreas development.     

抗衰老的GDF11生物学特征与功能表现

生长分化因子11（growth differentiation factor-11, GDF11）是转化生长因子β(transforming growth factor-β, TGF-β) 超家族中骨形态发生蛋白（bone morphogenetic proteins,BMPs）亚家族中的一个重要成员,在哺乳动物的骨骼、肾等多种器官组织上均有表达,且在胚胎发育、骨骼和肌肉形成等方面起着重要作用。近年研究发现,GDF11与哺乳动物的抗衰老作用联系越来越紧密。本文整理国内外关于GDF11与衰老关系的研究,对 GDF11生物学基础以及对动物心脏的衰老、认知能力以及骨骼肌肉等方面的影响进行了综述。我们认为,GDF11作为一种新的细胞因子,可以调控多种下游信号通路,其作用的方式及影响还有待研究。GDF11研究可为在抗衰老以及与衰老相关疾病的治疗提供一定的理论基础。     

Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-β signaling

We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-β signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-β signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-β1 levels, phosphorylation of Smad2/3, and increased expression of p15^INK4B and p57^KIP2. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-β1 induced dephosphorylation of Smad2/3, and diminished the level of p15^INK4B and p57^KIP2. Moreover, Bmi-1 expression led to the inhibition of TGF-β-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15^INK4B, and p57^KIP2. In addition, an exposure of senescent NHOK to TGF-β receptor I kinase inhibitor or anti-TGF-β antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-β signaling pathway in NHOK.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.