To delineate specific developmental roles of transforming growth factor fPI (TGF-P1) we Five distinct TGF-P3 genes have been identified in vertebrates and three of these (TGF-f31, TGF-p2, and TGF-(33) are expressed in mammals. Each of the three isoforms has been highly conserved throughout evolution, suggesting specific roles for each (1, 2). These three isoforms share a high degree of amino acid sequence homology in the mature domain, are often coexpressed and colocalized, and have qualitatively similar actions on tissue culture cells (1, 2). Therefore, it has been difficult to define the precise biological role ofindividual TGF-3 isoforms. To delineate and define the specific in vivo role of TGF-f31, we disrupted the murine TGF-,B1 gene in embryonic stem (ES) cells by homologous recombination (for reviews, see refs. 10 and 11). The targeted cells were subsequently used to generate mice with a loss-of-function mutation at the TGF-381 locus. Although the TGF-f31 null mutation in the homozygous state causes some intrauterine lethality, more than one-third of the fetuses develop to term and appear clinically normal at birth. After 2 weeks these mice develop a wasting syndrome and die -1-2 weeks later. Massive inflammatory lesions are seen in many organs, including the lungs (vasculitis, perivascular cuffing, and interstitial pneumonia) and heart (endocarditis and myocarditis), suggesting an uncontrolled inflammatory response that leads to premature death.MATERIALS AND METHODS Constructs. A 5.7-kb Bgl II genomic fragment containing the first two exons of TGF-,31 from the previously described clone pB2 (12) was subcloned into the modified Bluescript KS vector (Stratagene) in which the Asp 718 site was converted to a Sfi I site to generate pB2-3. A 560-bp sequence spanning part of the first exon (154 bp of coding sequence) and intron was deleted following Asp 718 digestion of pB2-3, and the phosphoglycerate kinase (PGK)-neomycin resistance (neo) gene (13) was inserted. The targeting vector, pTC-1, also contained a PGK-driven herpes simplex virus thymidine kinase gene (PGK-HSVtk) at the 3' end (see Fig. 1). Both marker genes also contain PGK poly(A) signals (13).ES Cell Culture, Transfection, and Selection. The CCE ES cell line (a generous gift from E. Robertson, Columbia University) was cultured on mitomycin-treated STO feeder layers in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (HyClone) and 10%o newborn calf serum (GIBCO) as described (14). ES cells were grown to 70% confluency, trypsinized, and resuspended in phosphatebuffered saline (PBS; Ca2+ and Mg2+ free) at 107 cells per ml.

Transforming growth factor-beta (TGFbeta) regulates the activation state of the endothelium via two opposing type I receptor/Smad pathways. Activin receptor-like kinase-1 (ALK1) induces Smad1/5 phosphorylation, leading to an increase in endothelial cell proliferation and migration, while ALK5 promotes Smad2/3 activation and inhibits both processes. Here, we report that ALK5 is important for TGFbeta/ALK1 signaling; endothelial cells lacking ALK5 are deficient in TGFbeta/ALK1-induced responses. More specifically, we show that ALK5 mediates a TGFbeta-dependent recruitment of ALK1 into a TGFbeta receptor complex and that the ALK5 kinase activity is required for optimal ALK1 activation. TGFbeta type II receptor is also required for ALK1 activation by TGFbeta. Interestingly, ALK1 not only induces a biological response opposite to that of ALK5 but also directly antagonizes ALK5/Smad signaling.

SummaryDiamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, non-classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence-based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.

Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-β type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-β/Smad signaling in HSC maintenance. TGF-β is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-β. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-β-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-β.