Mice lacking TGF-β3 exhibit an incompletely penetrant failure of the palatal shelves to fuse leading to cleft palate. The defect appears to result from impaired adhesion of the apposing medial edge epithelia of the palatal shelves and subsequent elimination of the mid-line epithelial seam. No craniofacial abnormalities were observed. This result demonstrates that TGF-β3 affects palatal shelf fusion by an intrinsic, primary mechanism rather than by effects secondary to craniofacial defects.Members of the transforming growth factor-β (TGF-β) gene family have biological activities that control cell proliferation, migration and differentiation, regulation of extracellular matrix deposition and epithelial-mesenchymal transformation [1][2][3] . Mammals contain three highly conserved isoforms of TGF-β, termed TGF-β1, TGF-β2 and TGF-β3, which display distinctive, although at times overlapping, spatial and temporal expression patterns [4][5][6] . Previous studies suggested that TGF-β3 may play a crucial role during palatogenesis 7-9 , Meckel's cartilage formation 10 , cardiac morphogenesis 11 , mammary gland development 12 and wound healing 13 . Other tissues expressing TGF-β3 in significant levels are cartilage, bone, brain and lung [4][5][6]14 .In mammalian palatogenesis apposition of the palatal shelves, adhesion of the medial edge epithelia (MEE) and subsequent elimination of the epithelial seam lead to a seamless mesenchymal shelf separating the oral and nasal cavities 15 . In vitro organ culture studies indicate that TGF-β1 and TGF-β2 accelerate palatal shelf fusion 16,17 and that antisense oligodeoxynucleotides or neutralizing antibodies to TGF-β3, but not to TGF-β1 or TGF-β2, block the fusion process 9 . We have now created mice deficient in TGF-β3, and show that this factor has a role in palatal shelf fusion by means of an intrinsic, primary mechanism and not by effects secondary to craniofacial morphometrics. A comparison of this defect to the inflammatory disorder of TGF-β1-deficient mice [18][19][20][21] Mutation of TGF-β3 in ES cellsThe TGF-β3 gene was mutated in ES cells (Fig. 1a) by replacing exon 6, the first full exon encoding sequences of the active domain of the protein, with the neomycin-resistance gene from pMC1neo 22 . Diagnostic Southern blots of the clone I98 indicated that the locus was successfully targeted; the proper genomic regions flanking both sides of the target site remained intact (Fig. 1b). Probing with a neo-gene probe indicated that there was only one integration site (not shown). Consequently, only the TGF-β3 locus has been disrupted. RT-PCR analysis of whole 11.5- (Fig. 1c) and 15.5-day embryos (not shown) indicated no TGF-β3 expression in homozygous mutant embryos, and revealed no significant change in the expression of TGF-β1 or TGF-β2 in the absence of TGF-β3. Cleft palate in TGF-β3 null mutantsThe targeted ES cell clone I98 was used to produce chi-maeric mice, which were mated with CF-1, C57BL/6 or 129/Sv mice. Heterozygous offspring showed no apparent phenotype. Interc...

The efficiency of producing timed pregnant or pseudopregnant mice can be increased by identifying those in proestrus or estrus. Visual observation of the vagina is the quickest method, requires no special equipment, and is best used when only proestrus or estrus stages need to be identified. Strain to strain differences, especially in coat color can make it difficult to determine the stage of the estrous cycle accurately by visual observation. Presented here are a series of images of the vaginal opening at each stage of the estrous cycle for 3 mouse strains of different coat colors: black (C57BL/6J), agouti (CByB6F1/J) and albino (BALB/cByJ). When all 4 stages (proestrus, estrus, metestrus, and diestrus) need to be identified, vaginal cytology is regarded as the most accurate method. An identification tool is presented to aid the user in determining the stage of estrous when using vaginal cytology. These images and descriptions are an excellent resource for learning how to determine the stage of the estrous cycle by visual observation or vaginal cytology.

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction proffle of marker genes for primitive endoderm (collagen a IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL.6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and ILH11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.

A set of 1638 informative SNP markers easily assayed by the Amplifluor genotyping system were tested in 102 mouse strains, including the majority of the common and wild-derived inbred strains available from The Jackson Laboratory. Selected from publicly available databases, the markers are on average ∼1.5 Mb apart and, whenever possible, represent the rare allele in at least two strains. Amplifluor assays were developed for each marker and performed on two independent DNA samples from each strain. The mean number of polymorphisms between strains was 608±136 SD. Several tests indicate that the markers provide an effective system for performing genome scans and quantitative trait loci analyses in all but the most closely related strains. Additionally, the markers revealed several subtle differences between closely related mouse strains, including the groups of several 129, BALB, C3H, C57, and DBA strains, and a group of wild-derived inbred strains representing several Mus musculus subspecies. Applying a neighbor-joining method to the data, we constructed a mouse strain family tree, which in most cases confirmed existing genealogies.

Xenopus in vitro studies have implicated both transforming growth factor ␤ (TGF-␤) and fibroblast growth factor (FGF) families in mesoderm induction. Although members of both families are present during mouse mesoderm formation, there is little evidence for their functional role in mesoderm induction. We show that mouse embryonic stem cells, which resemble primitive ectoderm, can differentiate to mesoderm in vitro in a chemically defined medium (CDM) in the absence of fetal bovine serum. In CDM, this differentiation is responsive to TGF-␤ family members in a concentration-dependent manner, with activin A mediating the formation of dorsoanterior-like mesoderm and bone morphogenetic protein 4 mediating the formation of ventral mesoderm, including hematopoietic precursors. These effects are not observed in CDM alone or when TGF-␤1, -␤2, or -␤3, acid FGF, or basic FGF is added individually to CDM. In vivo, at day 6.5 of mouse development, activin ␤A RNA is detectable in the decidua and bone morphogenetic protein 4 RNA is detectable in the egg cylinder. Together, our data strongly implicate the TGF-␤ family in mammalian mesoderm development and hematopoietic cell formation.

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a singledominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a ''minimal essential MHC'' for chickens, in strong contrast to typical mammals.antigen presentation ͉ avian ͉ essential ͉ evolution ͉ minimal

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.

Development of definitive (fetal liver-derived) red cells is blocked by a targeted mutation in the gene encoding the transcription factor GATA-1. We used in vitro differentiation of GATA-1- mouse embryonic stem (ES) cells to reveal a requirement for GATA-1 during primitive (yolk sac-derived) erythropoiesis and to establish a rescue assay. We show that the block to development includes primitive, as well as definitive, erythroid cells and is complete at the level of globin RNA expression; that the introduction of a normal GATA-1 gene restores developmental potential both in vivo and in vitro; and that efficient rescue is dependent on a putative autoregulatory GATA-motif in the distal promoter. Use of in vitro differentiated ES cells bridges a gap between conventional approaches to gene function in cell lines and analysis of loss of function mutations in the whole animal.