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.

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. CdkS purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of CdkS(-/-) mice through gene targeting and their phenotypic analysis.CdkS(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of CdkS(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.

The molecular mechanisms directing the development of 'natural' CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) in the thymus are not thoroughly understood. We show here that conditional deletion of transforming growth factor-beta receptor I (TbetaRI) in T cells blocked the appearance of CD4+CD25+Foxp3+ thymocytes at postnatal days 3-5. Paradoxically, however, beginning 1 week after birth, the same TbetaRI-mutant mice showed accelerated expansion of thymic CD4+CD25+Foxp3+ populations. This rapid recovery of Foxp3+ thymocytes was attributable mainly to overproduction of and heightened responsiveness to interleukin 2, as genetic ablation of interleukin 2 in TbetaRI-mutant mice resulted in a complete absence of CD4+CD25+Foxp3+ cells from the thymus and periphery. Thus, transforming growth factor-beta signaling is critical to the thymic development of natural CD4+CD25+Foxp3+ T(reg) cells.