Activin/TGFbeta and BMP crosstalk determines digit chondrogenesis

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFβ signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFβ and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.     

TGFbeta superfamily signaling: notes from the desert

The TGFbeta pathways play crucial roles in many developmental events, as well as contributing to many disease states. To provide a venue for both signaling and developmental research on TGFbeta, a FASEB-sponsored bi-annual meeting was initiated six years ago, the fourth of which was organized by Caroline Hill and Michael O'Connor and took place this July in Tucson, Arizona. The meeting highlighted major advances in our understanding of the structural and biochemical aspects of TGFbeta superfamily signaling, its intersection with other pathways, and its contribution to disease.     

What MAN1 does to the Smads. TGFbeta/BMP signaling and the nuclear envelope

The inner nuclear membrane protein MAN1 has been identified as an important factor in transforming growth factor beta/bone morphogenic protein (TGFbeta/BMP) signaling. Loss of MAN1 results in three autosomal dominant diseases in humans; all three characterized by increased bone density. Xenopus embryos lacking MAN1 develop severe morphological defects. Both in humans and in Xenopus embryos the defects originate from deregulation of TGFbeta/BMP signaling. Several independent studies have shown that MAN1 is antagonizing TGFbeta/BMP signaling through binding to regulatory Smads. Here, recent progress in understanding MAN1 functions is summarized and a model for MAN1-dependent regulation of TGFbeta/BMP signaling is proposed.     

Contributions by members of the TGFbeta superfamily to lens development

Members of the TGFbeta superfamily of growth and differentiation factors, including the TGFbeta, BMP, activin and nodal families, play important signaling roles throughout development. This paper summarizes some of the functions of these ligands in lens development. Targeted deletion of the genes encoding one of the BMP receptors, Alk3 (BMP receptor-1A), showed that signaling through this receptor is essential for normal lens development. Lenses lacking Alk3 were smaller than normal, with thin epithelial layers. The fiber cells of Alk3 null lenses became vacuolated and degenerated within the first week after birth. Lenses lacking Alk3 function were surrounded by abnormal mesenchymal cells, suggesting that the lenses provided inappropriate signals to surrounding tissues. Lens epithelial and fiber cells contained endosomes that were associated with activated (phosphorylated) SMAD1 and SMAD2. Endosomal localization of pSMAD1 was reduced in the absence of Alk3 signaling. The presence of pSMAD2 in lens fiber cell nuclei and the observation that the activin antagonist follistatin inhibited lens cell elongation suggested that an activin-like molecule participates in lens fiber cell differentiation. Lenses deficient in type II TGFbeta receptors were clear and had fiber cells of normal morphology. This suggests that TGFbeta signaling is not essential for the normal differentiation of lens fiber cells. The targeted deletion of single or multiple receptors of the TGFbeta superfamily in the lens should further characterize the role of these signaling molecules in lens development. This approach may also provide a useful way to define the downstream pathways that are activated by these receptors during the development of the lens and other tissues.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Regulation of human erythropoiesis by activin A,BMP2, and BMP4, members of the TGFbeta family

Activin A, BMP2, and BMP4, members of the TGFbeta family, have been implicated in the regulation of hematopoiesis. Here we explore and compare, for the first time in human primary cells, the role of activin A, BMP2, and BMP4 during erythropoiesis. Using in vitro erythroid differentiation of CD34(+) primary cells, we obtained the main stages of early erythropoiesis, characterized at the molecular, biochemical, and functional levels. Our results indicate that BMP2 acts on early erythroid cells and activin A on a more differentiated population. We report an insight into the mechanism of commitment of erythropoiesis by activin A and BMP2 involving two key events, increase in EPO-R and decrease in GATA2 expression. Simultaneous addition of activin A with BMP molecules suggests that BMP2 and BMP4 differently affect activin A induction of erythropoiesis. Follistatin and FLRG proteins downmodulate the effects of activin A and BMP2 on erythroid maturation.     

TGFbeta superfamily signals are required for morphogenesis of the kidney mesenchyme progenitor population

The TGFbeta superfamily plays diverse and essential roles in kidney development. Gdf11 and Bmp4 are essential for outgrowth and positioning of the ureteric bud, the inducer of metanephric mesenchyme. During nephrogenesis, Bmp7 is required for renewal of the mesenchyme progenitor population. Additionally, in vitro studies demonstrate inhibitory effects of BMPs and TGFbetas on collecting duct branching and growth. Here, we explore the predicted models of TGFbeta superfamily function by cell-specific inactivation of Smad4, a key mediator of TGFbeta signaling. Using a HoxB7cre transgene expressed in ureteric bud and collecting duct, we find that development of the collecting duct is Smad4 independent. By contrast, removal of Smad4 in nephrogenic mesenchyme using the Bmp7(cre/+) allele leads to disorganization of the nephrogenic mesenchyme and impairment of mesenchyme induction. Smad4-deficient metanephric mesenchyme does not display defects in inducibility in LiCl or spinal cord induction assays. However, in situ hybridization and lineage analysis of Smad4 null mesenchyme cells at E11.5 show that the nephrogenic mesenchyme does not aggregate tightly around the ureteric bud tips, but remains loosely associated, embedded within a population of cells expressing markers of both nephrogenic mesenchyme and peripheral stroma. We conclude that the failure of recruitment of nephrogenic mesenchyme leaves a primitive population of mesenchyme at the periphery of the kidney. This population is gradually depleted, and by E16.5 the periphery is composed of cells of stromal phenotype. This study uncovers a novel role for TGFbeta superfamily signaling in the recruitment and/or organization of the nephrogenic mesenchyme at early time-points of kidney development. Additionally, we present conclusive genetic lineage mapping of the collecting duct and nephrogenic mesenchyme.     

Cooperativity of thymopoietin 32-36 (the active site) and thymopoietin 38-45 in receptor binding

Thymopoietin is a 49 amino acid polypeptide hormone of the thymus whose biological activity is reproduced by the synthetic pentapeptide thymopentin, corresponding to amino acids 32-36. Thymopentin requires the addition of an octapeptide corresponding to thymopoietin 38-45 for full competition with native thymopoietin in a radioreceptor assay with receptor derived from the human T-cell line CEM. Thus thymopoietin appears to bind to its receptor on T-cells by two regions (32-36 and 38-45). Thymopentin alone is biologically active and induces elevations of intracellular cyclic GMP. Whilst occupancy of the adjacent site by thymopoietin 37-45 does not of itself cause an elevation of intracellular cyclic GMP this peptide is not biologically silent as it does enhance the potency of thymopentin.     

Mapping of epitopes in discoidin domain receptor 1 critical for collagen binding

The binding and activation of the discoidin domain receptor 1 by collagen has led to the conclusion that proteins from the extracellular matrix can directly induce receptor tyrosine kinase-mediated signaling cascades. A region in the extracellular domain of DDR1 homologous to the Dictyostelium discoideum protein discoidin-I is also present in the secreted human protein RS1. Mutations in RS1 cause retinoschisis, a genetic disorder characterized by ablation of the retina. By introducing point mutations into the discoidin domain of DDR1 at positions homologous to the retinoschisis mutations, ligand binding epitopes in the discoidin domain of DDR1 were mapped. Surprisingly, some residues only affected receptor phosphorylation, whereas others influenced both collagen-binding and receptor activation. Furthermore, two truncated DDR1 variants, lacking either the discoidin domain or the stalk region between the discoidin and transmembrane domain, were generated. We showed that (i) the discoidin domain was necessary and sufficient for collagen binding, (ii) only the region between discoidin and transmembrane domain was glycosylated, and (iii) the entire extracellular domain was essential for transmembrane signaling. Using these results, we were able to predict key sites in the collagen-binding epitope of DDR1 and to suggest a potential mechanism of signaling.     

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-beta (TGFbeta) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, we undertook a biochemical approach to establish the ligand binding repertoire and downstream signaling pathway. We observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor thickveins and saxophone to activate a BMP-like signaling pathway. Further we demonstrated that both myoglianin and its most closely related mammalian ligand, myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFbeta/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFbeta-like signaling pathways. Given that myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate myoglianin-dependent signals in the nervous system.     

BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein

Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.