Analysis Of Chimeras Research Articles

Evolution of the human immunodeficiency virus type 1 envelope during infection reveals molecular corollaries of specificity for coreceptor utilization and AIDS pathogenesis.

The evolution of human immunodeficiency virus type 1 infection is associated with a shift in the target cell population, driven by variability in coreceptor utilization resulting from diversity in env. To elucidate the potential consequences of these changes for Env-mediated fusion over the course of AIDS, we examined the biological properties of serial viral isolates and determined coreceptor utilization by the products of env cloned from two individuals, followed from the detection of seroconversion throughout the course of their infection. One had a typical course, and the other had an accelerated progression. Early isolates were non-syncytium inducing, and the corresponding Env exclusively utilized CCR5, whereas Env from late phases of infection showed restricted utilization of CXCR4 in both patients. Env from subject SC24, who had a standard progression, demonstrated multitropism, manifested by utilization of CCR3, CXCR4, and CCR5 in the intervening period. In contrast, Env from patient SC51, who experienced early conversion to the syncytium-inducing phenotype, developed dualtropic coreceptor utilization of CCR5 and CXCR4. Genetic analysis of env from each isolate revealed that those with an X4 phenotype formed a distinct subcluster within each subject. Analysis of chimeras constructed from R5 and multispecific env from patient SC24 demonstrated that while the V3 domain played a dominant role in determining coreceptor utilization, sequences in the V4-V5 region also contributed to the latter phenotype. Immunoprecipitation experiments confirmed that the hybrid Env proteins were expressed at similar levels. These experiments demonstrate that progression from the R5 to X4 phenotype may occur through a multi- or dual-tropic intermediate and that multiple domains contribute to this process.

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

The structure-function relationship of functionally distinct but structurally similar hexose transporters from Trypanosoma congolense.

We have previously characterized, in Trypanosoma brucei, a multigene family encoding two developmentally regulated glucose transporters that are 80% identical at the amino-acid level. We report here the characterization of the homologous glucose transporters (TcoHT1 and TcoHT2) in Trypanosoma congolense, an African trypanosome responsible for disease in domestic animals. Both TcoHT isoforms, which are 92.4% identical, are encoded by a single cluster of genes containing two copies of TcoHT1 and three copies of TcoHT2 arranged alternately. Northern blot analysis revealed that TcoHT2 is expressed in all of the adaptive forms, while mRNA encoding TcoHT1 is only present in the metacyclic and bloodstream forms of T. congolense. When transfected with the TcoHT2 gene, Chinese Hamster Ovary cells express a hexose transporter with properties similar to those of the T. congolense procyclic forms (Km D-glucose = 41 microM versus 64 microM). In contrast to TcoHT2, TcoHT1 expressed in the Chinese hamster ovary cells appeared to be a relatively low affinity glucose transporter (Ki D-glucose = 0.8 mM). To determine the region(s) involved in the different apparent affinities for glucose, a chimera analysis was undertaken on the TcoHT isoforms. This study shows that amino-acid residues important for D-glucose recognition are located in the central region (between transmembrane domains 3 and 7) and in the C-terminal intracellular domain of TcoHT2. Site directed mutagenesis identified Ser193 located within transmembrane helix 4 as a key residue in relaxing the apparent affinity of TcoHT1 for glucose.

MesP1 and MesP2 are essential for the development of cardiac mesoderm.

The transcription factors, MesP1 and MesP2, sharing an almost identical bHLH motif, have an overlapping expression pattern during gastrulation and somitogenesis. Inactivation of the Mesp1 gene results in abnormal heart morphogenesis due to defective migration of heart precursor cells, but somitogenesis is not disrupted because of normal expression of the Mesp2 gene. To understand the cooperative functions of MesP1 and MesP2, either a deletion or sequential gene targeting strategy was employed to inactivate both genes. The double-knockout (dKO) embryos died around 9.5 days postcoitum (dpc) without developing any posterior structures such as heart, somites or gut. The major defect in this double-knockout embryo was the apparent lack of any mesodermal layer between the endoderm and ectoderm. The abnormal accumulation of cells in the primitive streak indicates a defect in the migratory activity of mesodermal cells. Molecular markers employed to characterize the phenotype revealed a lack of the cranio-cardiac and paraxial mesoderm. However, the axial mesoderm, as indicated by brachyury (T) expression, was initially generated but anterior extension was halted after 8.5 dpc. Interestingly, a headfold-like structure developed with right anterior-posterior polarity; however, the embryos lacked any posterior neural properties. The persistent and widely distributed expression of Cerberus-like-1(Cer1), Lim1 and Otx2 in the anterior endoderm might be responsible for the maintenance of anterior neural marker expression. We also performed a chimera analysis to further study the functions of MesP1 and MesP2 in the development of mesodermal derivatives. In the chimeric embryos, dKO cells were scarcely observed in the anterior-cephalic and heart mesoderm, but they did contribute to the formation of the somites, notochord and gut. These results strongly indicate that the defect in the cranial-cardiac mesoderm is cell-autonomous, whereas the defect in the paraxial mesoderm is a non-cell-autonomous secondary consequence.

Amino Acid Residues Conferring Ligand Binding Properties of Prostaglandin I and Prostaglandin D Receptors: IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS

Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.

Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length.

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants. The NodC proteins of Mesorhizobium loti and Sinorizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli. The oligosaccharides produced were analyzed using thin-layer chromatography. The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the characteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids; exchange of only this C-terminal region is sufficient to completely convert the M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer. However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length.

Intestinal inflammation observed in IL-2R/IL-2 mutant mice is associated with impaired intestinal T lymphopoiesis

Background & Aims: Although interleukin (IL)-2 −/− and IL-2Rα −/− mice develop inflammatory bowel disease, IL-2Rβ −/− animals are apparently free of gut pathology. Intraintestinal T lymphopoiesis is reported to be impaired in IL-2Rβ −/− mice; we have determined whether this characteristic correlated with the apparent resistance of this mutant strain to intestinal inflammation. This led us to reassess intraintestinal T lymphopoiesis in these 3 mutant strains. Methods: Intestinal histology and intraintestinal T lymphopoiesis were analyzed in unmanipulated mutant mice and in athymic and euthymic radiation chimeras reconstituted with bone marrow derived from IL-2 −/−, IL-2Rα −/−, and IL-2Rβ −/− donors. Results: Intraintestinal T lymphopoiesis was ablated in the 3 mutant strains and was associated with cryptopatch abnormalities. The intestinal mucosa of mice reconstituted with lymphocytes from IL-2Rβ −/− mice exhibited lesions of both the small and large bowel similar to those observed in the early stages of human gluten enteropathy and acute ulcerative colitis, respectively. Analysis of euthymic and athymic bone marrow radiation chimeras indicated that T cells located in the intestinal mucosa of unmanipulated IL-2 −/−, IL-2Rα −/−, and IL-2Rβ −/− mice are of thymic origin. Conclusions: Null mutations at IL-2/IL-2Rα and β loci differentially affect intraintestinal and intrathymic T lymphopoiesis. These conditions are associated with lesions of intestinal inflammation that are mediated by thymus-derived T cells. GASTROENTEROLOGY 2000;118:880-891

Medial Golgi but Not Late Golgi Glycosyltransferases Exist as High Molecular Weight Complexes: ROLE OF LUMINAL DOMAIN IN COMPLEX FORMATION AND LOCALIZATION

To investigate the organization of Golgi glycosyltransferases and their mechanism of localization, we have compared the properties of a number of medial and late acting Golgi enzymes. The medial Golgi enzymes, N-acetylglucosaminyltransferase I and II (GnTI and GnTII) required high salt for solubilization and migrated as high molecular weight complexes on sucrose density gradients. In contrast, the late acting Golgi enzymes, beta1,4-galactosyltransferase and alpha1, 2-fucosyltransferase, were readily solubilized in low salt and migrated as monomers/dimers by sucrose density gradient centrifugation. Analysis of membrane-bound GnTI chimeras indicates that the formation of high molecular weight complexes does not require the transmembrane domain and cytoplasmic tail sequences of GnTI. Furthermore, a soluble form of GnTI, containing the stem region and catalytic domain, accumulated in the Golgi prior to secretion, in contrast to beta1,4-galactosyltransferase. Soluble GnTI, which also associated with high molecular weight complexes, was comparable with membrane-bound GnTI in its ability to glycosylate newly synthesized glycoproteins in vivo. Mutation of charged residues within the stem region of GnTI, known to be important for "kin recognition", had no effect on the efficiency of Golgi localization, the inclusion into high molecular weight complexes, nor functional activity in vivo. The differences in behavior between the medial and late acting Golgi enzymes may contribute to their differential localization and their ability to glycosylate efficiently in the correct Golgi subcompartment.

Molecular determinants that mediate selective activation of p38 MAP kinase isoforms.

The p38 mitogen-activated protein kinase (MAPK) group is represented by four isoforms in mammals (p38alpha, p38beta2, p38gamma and p38delta). These p38 MAPK isoforms appear to mediate distinct functions in vivo due, in part, to differences in substrate phosphorylation by individual p38 MAPKs and also to selective activation by MAPK kinases (MAPKKs). Here we report the identification of two factors that contribute to the specificity of p38 MAPK activation. One mechanism of specificity is the selective formation of functional complexes between MAPKK and different p38 MAPKs. The formation of these complexes requires the presence of a MAPK docking site in the N-terminus of the MAPKK. The second mechanism that confers signaling specificity is the selective recognition of the activation loop (T-loop) of p38 MAPK isoforms. Together, these processes provide a mechanism that enables the selective activation of p38 MAPK in response to activated MAPKK.

Chimera analysis of troponin I domains that influence Ca(2+)-activated myofilament tension in adult cardiac myocytes.

The goal of this study was to investigate isoform-specific functional domains of the inhibitory troponin subunit, troponin I (TnI), as it functions within the intact myofilaments of adult cardiac myocytes. Adenovirus-mediated gene transfer was used to deliver and express a TnI chimera composed of the amino terminus of cardiac TnI (cTnI) and the carboxy terminus of slow skeletal TnI (ssTnI) in adult rat cardiac myocytes. The TnI chimera, designated N-card/slow-C TnI, was expressed and incorporated into myofilaments after gene transfer, without detectable changes in contractile protein stoichiometry or sarcomere architecture. Interestingly, force at submaximal Ca(2+) levels was markedly elevated in single permeabilized myocytes expressing the N-card/slow-C TnI chimera relative to force generated in adult myocytes expressing ssTnI or cTnI. Based on these results, a hierarchy of myofilament Ca(2+) sensitivity is emerging by use of TnI chimera analysis, with the order of sensitivity being N-card/slow-C TnI>>ssTnI>>cTnI. These results also strongly suggest that independent isoform-specific domains in both the amino and carboxy portions of TnI influence myofilament Ca(2+) sensitivity. In additional studies carried out under pathophysiological ionic conditions (pH 6.2), the dramatic acidosis-induced decrease in myofilament Ca(2+) sensitivity observed in myocytes expressing cTnI was blunted in myocytes expressing N-card/slow-C TnI in a manner similar to that in ssTnI-expressing myocytes. These results demonstrate that there is a pH-sensitive domain residing in the carboxy-terminal portion of TnI. The dissection of isoform-specific functional domains under physiological and acidic pH conditions demonstrates the utility of TnI chimeras for analysis of TnI function and provides important insights into the overall function of TnI within the intact myofilament of adult cardiac myocytes.

Genomic analysis of ABO chimeras and mosaics using hematopoietic colony-derived DNA.

While there are many case reports dealing with ABO mosaicism and chimerism, there have been few attempts to determine the patient's genotype. Peripheral blood and buccal mucosa were obtained from three persons with ABO mosaicism or chimerism. DNA extracted from hematopoietic progenitor cell-derived colonies and from peripheral blood and buccal mucosa were analyzed by polymerase chain reaction-restriction fragment length polymorphism methods. In addition, analyses of short tandem repeat markers were carried out. Hematopoietic progenitor cell-derived DNA analysis revealed that, in two of the three persons there were 2 apparently distinct progenitor cell lineages whose percentages were close to those in the peripheral blood of the patients, as analyzed by flow cytometry; the exception was Subject 3, who had myelodysplastic syndrome (MDS). Short tandem repeat analysis showed that the former two subjects had two pairs of ABO alleles and the latter subject, with MDS, had loss of heterozygosity in some colony-derived DNA as well as blood DNA. The subjects without MDS had two distinct hematopoietic cell lineages that led to their ABO chimeric status. The subject with MDS was assumed to have an ABO mosaicism caused by the somatic deletion of the ABO gene in the hematopoietic progenitor cells.

Inhibition of Na(+)-K(+)-2Cl(-) cotransport by mercury.

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH(-)) groups. The inorganic form (Hg(2+)) is toxic to epithelial tissues and interacts with various transport proteins including the Na(+) pump and Cl(-) channels. In this study, we determined whether the Na(+)-K(+)-Cl(-) cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg(2+) on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg(2+) inhibits Na(+)-K(+)-Cl(-) cotransport, with inhibitor constant (K(i)) values of 25 microM for the shark carrier (sNKCC1) and 43 microM for the human carrier. In further studies, we took advantage of species differences in Hg(2+) affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697-->Leu) reveals that transmembrane domain 11 plays an essential role in Hg(2+) binding. We also show that modification of additional SH(-) groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

Heterodimerization of the  and β Chains of the Interleukin-3 (IL-3) Receptor Is Necessary and Sufficient for IL-3–Induced Mitogenesis

The high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3–binding subunit (IL-3) and a larger β chain—βc, or, in the mouse, βc or its close relative βIL-3. There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of IL-3 and βIL-3, but is, rather, the formation of a β-β homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from IL-3, βc or βIL-3, and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of IL-3 and βIL-3. Homodimers of either IL-3 or βIL-3, alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of IL-3 and βIL-3.

Heterodimerization of the α and β Chains of the Interleukin-3 (IL-3) Receptor Is Necessary and Sufficient for IL-3–Induced Mitogenesis

AbstractThe high-affinity receptor for interleukin-3 (IL-3) is a complex of the IL-3–binding subunit (αIL-3) and a larger β chain—βc, or, in the mouse, βc or its close relative βIL-3. There is evidence that the critical event that initiates signaling is not the approximation of the cytoplasmic domains of αIL-3 and βIL-3, but is, rather, the formation of a β-β homodimer. Many of these studies involved the analyses of receptor chimeras where the cytoplasmic domains were derived from αIL-3, βc or βIL-3, and the extracellular domains were derived from other cytokine receptors, such as the erythropoietin receptor (EpoR). However, evidence that the EpoR may also associate with other receptors clouds the interpretation of these experiments. Therefore, we reevaluated the structure of the functional IL-3R using chimeric receptors with extracellular domains derived not from members of the cytokine-receptor family, but from CD8 or CD16. We show, by expression of these chimeras in Ba/F3 or CTLL-2 cells, that mitogenic signals were only generated by heterodimerization of the cytoplasmic domains of αIL-3 and βIL-3. Homodimers of either αIL-3 or βIL-3, alone or in combination, were nonfunctional. Furthermore, the ability of heterodimers to stimulate mitogenesis correlated with their ability to induce tyrosine phosphorylation of JAK-2. These data suggest that the physiological activation of the IL-3R involves the generation of simple heterodimers of αIL-3 and βIL-3.

The zebrafish diwanka gene controls an early step of motor growth cone migration.

During vertebrate embryogenesis different classes of motor axons exit the spinal cord and migrate on common axonal paths into the periphery. Surprisingly little is known about how this initial migration of spinal motor axons is controlled by external cues. Here, we show that the diwanka gene is required for growth cone migration of three identified subtypes of zebrafish primary motoneurons. In diwanka mutant embryos, motor growth cone migration within the spinal cord is unaffected but it is strongly impaired as motor axons enter their common path to the somites. Chimera analysis shows that diwanka gene activity is required in a small set of myotomal cells, called adaxial cells. We identified a subset of the adaxial cells to be sufficient to rescue the diwanka motor axon defect. Moreover, we show that this subset of adaxial cells delineates the common axonal path prior to axonogenesis, and we show that interactions between these adaxial cells and motor growth cones are likely to be transient. The studies demonstrate that a distinct population of myotomal cells plays a pivotal role in the early migration of zebrafish motor axons and identify the diwanka gene as a somite-derived cue required to establish an axonal path from the spinal cord to the somites.

Modulation of Human Heat Shock Factor Trimerization by the Linker Domain

Heat shock transcription factors (HSFs) are stress-responsive proteins that activate the expression of heat shock genes and are highly conserved from bakers' yeast to humans. Under basal conditions, the human HSF1 protein is maintained as an inactive monomer through intramolecular interactions between two coiled-coil domains and interactions with heat shock proteins; upon environmental, pharmacological, or physiological stress, HSF1 is converted to a homotrimer that binds to its cognate DNA binding site with high affinity. To dissect regions of HSF1 that make important contributions to the stability of the monomer under unstressed conditions, we have used functional complementation in bakers' yeast as a facile assay system. Whereas wild-type human HSF1 is restrained as an inactive monomer in yeast that is unable to substitute for the essential yeast HSF protein, mutations in the linker region between the DNA binding domain and the first coiled-coil allow HSF1 to homotrimerize and rescue the viability defect of a hsfDelta strain. Fine mapping by functional analysis of HSF1-HSF2 chimeras and point mutagenesis revealed that a small region in the amino-terminal portion of the HSF1 linker is required for maintenance of HSF1 in the monomeric state in both yeast and in transfected human 293 cells. Although linker regions in transcription factors are known to modulate DNA binding specificity, our studies suggest that the human HSF1 linker plays no role in determining HSF1 binding preferences in vivo but is a critical determinant in regulating the HSF1 monomer-trimer equilibrium.

The type I serine/threonine kinase receptor ActRIA (ALK2) is required for gastrulation of the mouse embryo.

ActRIA (or ALK2), one of the type I receptors of the transforming growth factor-beta (TGF-beta) superfamily, can bind both activin and bone morphogenetic proteins (BMPs) in conjunction with the activin and BMP type II receptors, respectively. In mice, ActRIA is expressed primarily in the extraembryonic visceral endoderm before gastrulation and later in both embryonic and extraembryonic cells during gastrulation. To elucidate its function in mouse development, we disrupted the transmembrane domain of ActRIA by gene targeting. We showed that embryos homozygous for the mutation were arrested at the early gastrulation stage, displaying abnormal visceral endoderm morphology and severe disruption of mesoderm formation. To determine in which germ layer ActRIA functions during gastrulation, we performed reciprocal chimera analyses. (1) Homozygous mutant ES cells injected into wild-type blastocysts were able to contribute to all three definitive germ layers in chimeric embryos. However, a high contribution of mutant ES cells in chimeras disrupted normal development at the early somite stage. (2) Consistent with ActRIA expression in the extraembryonic cells, wild-type ES cells failed to rescue the gastrulation defect in chimeras in which the extraembryonic ectoderm and visceral endoderm were derived from homozygous mutant blastocysts. Furthermore, expression of HNF4, a key visceral endoderm-specific transcription regulatory factor, was significantly reduced in the mutant embryos. Together, our results indicate that ActRIA in extraembryonic cells plays a major role in early gastrulation, whereas ActRIA function is also required in embryonic tissues during later development in mice.

Functions of c-Jun in liver and heart development.

Mice lacking the AP-1 transcription factor c-Jun die around embryonic day E13.0 but little is known about the cell types affected as well as the cause of embryonic lethality. Here we show that a fraction of mutant E13.0 fetal livers exhibits extensive apoptosis of both hematopoietic cells and hepatoblasts, whereas the expression of 15 mRNAs, including those of albumin, keratin 18, hepatocyte nuclear factor 1, beta-globin, and erythropoietin, some of which are putative AP-1 target genes, is not affected. Apoptosis of hematopoietic cells in mutant livers is most likely not due to a cell-autonomous defect, since c-jun-/- fetal liver cells are able to reconstitute all hematopoietic compartments of lethally irradiated recipient mice. A developmental analysis of chimeras showed contribution of c-jun-/- ES cell derivatives to fetal, but not to adult livers, suggesting a role of c-Jun in hepatocyte turnover. This is in agreement with the reduced mitotic and increased apoptotic rates found in primary liver cell cultures derived from c-jun-/- fetuses. Furthermore, a novel function for c-Jun was found in heart development. The heart outflow tract of c-jun-/- fetuses show malformations that resemble the human disease of a truncus arteriosus persistens. Therefore, the lethality of c-jun mutant fetuses is most likely due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis.

The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo.

Signaling by members of the TGFbeta superfamily is thought to be transduced by Smad proteins. Here, we describe a zebrafish mutant in smad5, designated somitabun (sbn). The dominant maternal and zygotic effect of the sbntc24 mutation is caused by a change in a single amino acid in the L3 loop of Smad5 protein which transforms Smad5 into an antimorphic version, inhibiting wild-type Smad5 and related Smad proteins. sbn mutant embryos are strongly dorsalized, similarly to mutants in Bmp2b, its putative upstream signal. Double mutant analyses and RNA injection experiments show that sbn and bmp2b interact and that sbn acts downstream of Bmp2b signaling to mediate Bmp2b autoregulation during early dorsoventral (D-V) pattern formation. Comparison of early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos reveal three phases of D-V patterning: an early sbn- and bmp2b-independent phase when a coarse initial D-V pattern is set up, an intermediate sbn- and bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and a later sbn-independent phase during gastrulation when the Bmp2/4 gradient is interpreted and cell fates are specified.

Chimera Analysis Reveals That Fibroblasts and Endothelial Cells Require Platelet-Derived Growth Factor Receptorβ Expression for Participation in Reactive Connective Tissue Formation in Adults but Not during Development

The hypothesis that platelet-derived growth factor (PDGF) plays an important role in repair of connective tissue has been difficult to test experimentally, in part because the disruption of any of the PDGF ligand and receptor genes is embryonic lethal. We have developed a method that circumvents the embryonic lethality of the PDGF receptor (R)beta-/- genotype and minimizes the tendency of compensatory processes to mask the phenotype of gene disruption by comparing the behavior of wild-type and PDGFRbeta-/- cells within individual chimeric mice. This quantitative chimera analysis method has revealed that during development PDGFRbeta expression is important for all muscle lineages but not for fibroblast or endothelial lineages. Here we report that fibroblasts and endothelial cells, but not leukocytes, are dependent on PDGFRbeta expression during the formation of new connective tissue in and around sponges implanted under the skin. Even the 50% reduction in PDGFRbeta gene dosage in PDGFRbeta+/- cells reduces fibroblast and endothelial cell participation by 85%. These results demonstrate that the PDGFRbeta/PDGF B-chain system plays an important direct role in driving both fibroblast and endothelial cell participation in connective tissue repair, that cell behavior can be regulated by relatively small changes in PDGFRbeta expression, and that the functions served by PDGF in wound healing are different from the roles served during development.