Wild-type Chimeras Research Articles

Sorting Determinants in the Transmembrane Domain of p24 Proteins

Members of the p24 family of putative cargo receptors are proposed to contain retrograde and anterograde trafficking signals in their cytoplasmic domain to facilitate coat protein binding and cycling in the secretory pathway. We have analyzed the role of the transmembrane domain (TMD) of a p24 protein isolated from COPI-coated intra-Golgi transport vesicles. CD8-p24 chimeras were transiently expressed in COS7 cells and analyzed by immunofluorescence and pulse-chase experiments. The localization and transit of the wild-type chimera from the endoplasmic reticulum (ER) through the Golgi complex involved a glutamic acid residue and a conserved glutamine in the TMD. The TMD glutamic acid mediated the localization of the chimeras to the ER in the absence of the conserved glutamine. Efficient ER exit required the TMD glutamine and was further facilitated by a pair of phenylalanine residues in the cytoplasmic tail. TMD residues of p24 proteins may mediate the interaction with integral membrane proteins of the vesicle budding machinery to ensure p24 packaging into transport vesicles.

Changes in epitopes for thyroid-stimulating antibodies in Graves' disease sera during treatment of hyperthyroidism: therapeutic implications.

To determine whether there are changes in epitope recognition by stimulating TSH receptor antibodies (TSHRAbs) during treatment of hyperthyroidism and to evaluate the clinical relevance of such changes, we serially measured the activity of IgG preparations from 39 patients with Graves' disease over an 8-month period. To measure epitope changes of the stimulating TSHRAbs, we used Chinese hamster ovary (CHO) cells transfected with wild-type human TSHR (hTSHR) or TSHR chimeras with residues 90-165 (Mc2) substituted by equivalent residues of the rat LH/CG receptor. When initially examined, 37 of the 39 patients had significant stimulating TSHRAb activity measured with wild-type CHO-hTSHR cells. Serial measurements of stimulating TSHRAb activity in Mc2 chimera-transfected cells divided the 39 patients into three distinct groups. Thus, 10 patients (heterogeneous epitope group) exhibited low but significant activity in Mc2 chimera assays at the start of the study; 10 patients who were initially negative in Mc2 chimera assays remained negative (persistently homogeneous epitope group); and 19 patients who were initially negative in Mc2 chimera assays became transiently or persistently positive during treatment, despite a simultaneous decrease in TSHRAb activity measured with wild-type TSHR (changing epitope group). The functional stimulating TSHRAb epitope thus changed from residues 90-165 to residues outside this region in the last group, which comprises nearly two-thirds of the initially Mc2-negative patients (19 of 29) and one-half of all patients (19 of 39). Patients in the changing epitope group responded more quickly and to lower doses of methimazole than patients in the persistently homogeneous epitope group, behaving in this respect exactly as the patients in the heterogeneous epitope group. Additionally, although the decrease in stimulating TSHRAb activities during the 8-month treatment period was similar in the two groups, the thyrotropin binding inhibitor immunoglobulin (TBII) activities decreased more rapidly in patients in the persistently homogeneous epitope group than in patients in the changing epitope group (P < 0.05). There were no differences in initial stimulating TSHRAb or TBII activities, degree of hyperthyroidism, goiter size, or prior duration of symptoms between the persistently homogeneous epitope group and changing epitope group. In summation, we show that the epitopes of stimulating TSHRAbs in Graves' disease patients may change during their clinical course or treatment period, and that the change is from antibodies recognizing N-terminal TSHR residues 90-165 to antibodies recognizing other regions of the TSHR. We also show that the development of stimulating TSHRAbs with this heterogeneous epitope or their presence at the initial screening for disease activity seems to be associated with increased responsiveness to antithyroid drug therapy. We suggest, therefore, that Mc2 chimera assays may be useful to predict the response of patients to antithyroid drug therapy.

Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.

Subtypes of Anti-TSH Receptor Antibodies Classified by Various Assays Using CHO Cells Expressing Wild-Type or Chimeric Human TSH Receptor

To analyze the heterogeneity of anti-TSH receptor antibodies (TSHRAb), we measured serum TSH-binding inhibitory immunoglobulin (TBII), thyroid-stimulating antibody (TSAb), and thyroid stimulation blocking antibody (TSBAb) activities in 31 patients with positive TSHRAb, using CHO cells expressing wild-type TSHR (WT) or TSHR chimera (Mc2) wherein residues 90-165 were substituted by the LH/CG receptor. Using membranes from WT cells, we detected TBII activity in all 31 patients; 10 (32%), all with TSAb activity only, completely lost TBII activity using Mc2 membranes. TSAb activity was found in 26 sera using WT cells; 20 (77%) completely lost TSAb activity in Mc2 cells. Comparisons of TBII and TSAb activity in WT cells did not exhibit a strong positive correlation (r = 0.52). Of the 20 sera that completely lost TSAb activity in Mc2 cells, 10 retained some TBII activity in Mc2 cells. In each of the sera with retained TBII activity, TSAb activity was recovered in Mc2 cells using the conversion assay, which measures the conversion of a nonstimulating TSHRAb to a TSAb by the action of an anti-human IgG. Additionally, the TBII and conversion assay values in Mc2 cells exhibited a strong positive correlation (r = 0.86). Of the 31 sera, TSBAb was found in 7 samples, with no difference in WT and Mc2 cells. TBII activity was detected in all 7 sera with WT cells; TSAb activity in only 2. In the 5 sera with TSBAb but no TSAb activity, and with only a minimal or no decrease in TBII activity in Mc2 cell membranes, the in vitro conversion assay uncovered TSAb activity. Analyzing these data, we classify the sera into 5 groups containing multiple, different TSHR autoantibodies, including two different TSAbs, three different TBIIs, and one nonfunctional antibody. The heterogeneity of TBIIs as well as TSAbs provides a basis to explain the lack of correlation between TBII and TSAb activities in some past studies of Graves' sera.

The numerical matching of source and target populations in the CNS: the inferior olive to Purkinje cell projection

During a defined critical period of development, if the target of a neuronal population is removed, there is a massive decrease in the number of neurons that survive into adulthood. Previous studies have found that source neuron number is a strictly linear function of target size. The current work extends these observations to the inferior olive → Purkinje cell projection. Three distinct model systems have been used: (i) lurcher ↔ wild-type aggregation chimeras, (ii) staggerer ↔ wild-type chimeras and (iii) naturally occurring polymorphisms in Purkinje cell number found in different inbred mouse strains. Total neuron numbers were counted in the inferior olive and plotted as a function of the number of Purkinje cells in the contralateral cerebellar cortex. In lurcher mutants and chimeras, the relationship between these values is well described by a straight line. This suggests that, like the granule → Purkinje cell circuit, the olive → Purkinje cell circuit uses a linear algorithm to achieve a numerical balance. The results from the two other model systems were not as clear cut. In the staggerer chimeras, we found only a rough correlation between neuron and target numbers and in the inbred strains there was no discernible relationship at all. These findings indicate that in the final analysis, there are multiple factors involved in the determination of the number of olive cells surviving into adulthood. The potential contribution of sustaining collaterals and afferent inputs is discussed as well as the possible existence of different subcircuits of olivocerebellar connections, each with its own numerical matching function.

The numerical matching of source and target populations in the CNS: the inferior olive to Purkinje cell projection

During a defined critical period of development, if the target of a neuronal population is removed, there is a massive decrease in the number of neurons that survive into adulthood. Previous studies have found that source neuron number is a strictly linear function of target size. The current work extends these observations to the inferior olive → Purkinje cell projection. Three distinct model systems have been used: (i) lurcher ↔ wild-type aggregation chimeras, (ii) staggerer ↔ wild-type chimeras and (iii) naturally occurring polymorphisms in Purkinje cell number found in different inbred mouse strains. Total neuron numbers were counted in the inferior olive and plotted as a function of the number of Purkinje cells in the contralateral cerebellar cortex. In lurcher mutants and chimeras, the relationship between these values is well described by a straight line. This suggests that, like the granule → Purkinje cell circuit, the olive → Purkinje cell circuit uses a linear algorithm to achieve a numerical balance. The results from the two other model systems were not as clear cut. In the staggerer chimeras, we found only a rough correlation between neuron and target numbers and in the inbred strains there was no discernible relationship at all. These findings indicate that in the final analysis, there are multiple factors involved in the determination of the number of olive cells surviving into adulthood. The potential contribution of sustaining collaterals and afferent inputs is discussed as well as the possible existence of different subcircuits of olivocerebellar connections, each with its own numerical matching function.

Abnormal Purkinje cell dendrites in lurcher chimeric mice result from a deafferentation-induced atrophy.

Previous studies of Purkinje cell dendrites in lurcher<-->wild-type mouse chimeras (lurcher chimeras) have documented the surprising occurrence of unusual atrophic dendritic morphologies among the wild-type cells of the mosaic cerebella. We have hypothesized that these aberrant morphologies arise from a process of developmental deafferentation that is due to the unique loss of mutant Purkinje cells in these chimeras. These earlier studies left unanswered the question of whether the abnormal dendrites were the result of a blocked developmental process (agenesis) or regressive events that deform a previously well-developed dendritic arbor (atrophy). Using a set of simple morphometric measures, we now examine wild-type Purkinje cells in young lurcher chimeras. At postnatal day 20, normal Purkinje cell development is nearly but not fully complete. In lurcher chimeras, the morphologies of the wild-type Purkinje cell dendrites are similar to those in wild-type controls of the same age. This means that they are larger in height, width, and cross-section than their counterparts in adult lurcher chimeras. The younger cells exhibit almost none of the atrophic morphologies described in mature animals. We conclude that the aberrant morphologies found in adult lurcher chimeras arise from atrophy rather than through a failure in development. Furthermore, consideration of the details of the wild-type dendrites in the lurcher chimeras leads to the proposal that the height and width of the Purkinje cell dendritic tree are controlled by two independent mechanisms.

Intercellular signals downstream of endothelin receptor-B mediate colonization of the large intestine by enteric neuroblasts

Mice homozygous for the piebald lethal (sl) mutation, which have a complete deletion of endothelin receptor-B, fail to form ganglion cells in the distal large intestine and are nearly devoid of cutaneous melanocytes. These phenotypic features stem from incomplete colonization of the hindgut and skin by neural crest-derived neuroblasts and melanoblasts, respectively. We have used expression of a transgene, dopamine-beta-hydroxylase-nlacZ, to study colonization of the enteric nervous system in sl/sl embryos and sl/sl <--> wild-type chimeric mice. Enteric neuroblasts derived from the vagal neural crest colonize the developing foregut, midgut and distal small intestine of sl/sl embryos in a cranial-to-caudal manner indistinguishable from sl/+ or +/+ embryos. However, colonization of the large intestine is retarded and the distal large intestine is never colonized, a developmental defect identical to that observed in lethal spotted (endothelin-3 deficient) embryos. The coat pigmentation and relative distributions of mutant and wild-type ganglion cells in sl/sl <--> wild-type chimeras indicate that the defect associated with endothelin receptor-B gene deletion is not strictly neuroblast autonomous (independent of environmental factors). Instead, intercellular interactions downstream of the endothelin receptor-B mediate complete colonization of the skin and gut by neural crest cells.

Stunted morphologies of cerebellar Purkinje cells in lurcher and staggerer mice are cell-intrinsic effects of the mutant genes.

Purkinje cells in the neurological mutants lurcher and staggerer exhibit a number of abnormal properties; mutant<==>wild-type chimeras have shown that these properties are direct effects of the mutant gene. What has remained unexplored are the numerous dendritic abnormalities that the two mutant Purkinje cells exhibit. In staggerer, Purkinje cells have rudimentary, unbranched dendrites that lack tertiary branchlet spines. In lurcher, before the Purkinje cells die, their dendrites remain short and underdeveloped. To determine whether or not a system of healthy afferents (or other environmental factors) would alter either of these phenotypes, we examined young lurcher and adult staggerer mouse chimeras using Golgi impregnation. In postnatal day 20 (P20) lurcher chimeras, we found two distinct morphological classes of Purkinje cells. One, inferred to be wild type, had a dendritic structure similar to normal Purkinje cells in age-matched controls. The other consisted of cells with small somata, reduced dendritic arbors, and multiple dendritic processes, making them indistinguishable from Purkinje cells in P20 lurcher mutants. We also examined mature staggerer chimeras. We found no evidence that the stunted morphology of staggerer Purkinje cells is rescued in mosaic animals but observed numerous examples of medium to large neurons resembling atrophic Purkinje cells of staggerer mutants. These results suggest that the dendritic abnormalities described in both mutants reflect cell autonomous, developmental genetic blocks in the cytological maturation of the cerebellar Purkinje cell. The implication is that the action of the wild-type alleles at these two loci are required to execute a normal program of dendritic development.

A Theoretical and Experimental Examination of Cell Lineage Relationships among Cerebellar Purkinje Cells in the Mouse

In this paper, we continue our examination of the role of cell lineage in the development of the cerebellar Purkinje cell population of the mouse. The analysis of Purkinje cell lineage is based on counts of the number of wild-type Purkinje cells in +/ Lc ↔ wild-type chimeras. +/ Lc Purkinje cells undergo a cell autonomous degeneration early in post-natal development leaving variable numbers of wild-type Purkinje cells in chimeric animals. Using theoretical, statistical, and experimental approaches, we have tested various developmental models to account for the numerical development of Purkinje cell numbers in the +/ Lc ↔ wild-type chimeras. We have analyzed models based on the assumption that cell lineages are irrelevant to Purkinje cell development, as well as our own previous hypothesis that Purkinje cells descend from a small number of progenitor cells selected during the early stages of neurogenesis. The theoretical approach calculates the distributions of Purkinje cell numbers in hypothetical +/ Lc ↔ wild-type chimeras and inbred mice based on both clonal and nonclonal hypotheses of neuronal development. Variations of the model are compared with published cell counts from +/ Lc ↔ C3H/HeJ, +/ Lc ↔ C57BL/6J, +/ Lc ↔ AKR/J chimeras, and C3H/HeJ inbred mice. The statistical approach assesses the significance of the fits of the observed data with different variations of the model by Monte Carlo simulation techniques. The results of the comparison suggest that our observed data is more likely to be explained by a clonal model of development than by alternate models in which cell lineages play a minor role. Our experimental approach describes a new +/ Lc ↔ C3H/HeJ chimera in which all of the Purkinje cells (>7900) are found on one side of the brain. We have analyzed this chimera with respect to clonal and nonclonal models of Purkinje cell development. The extreme asymmetric distribution of Purkinje cells provides added support to the hypothesis that there is a small number of progenitor cells that generate Purkinje cells. Our findings lead to the conclusion that while not all alternate models of mammalian CNS development can be completely excluded, the early progenitor hypothesis is the most probable model of Purkinje cell development.

The surface envelope protein gene region of equine infectious anemia virus is not an important determinant of tropism in vitro.

Virulent, wild-type equine infectious anemia virus (EIAV) is restricted in one or more early steps in replication in equine skin fibroblast cells compared with cell culture-adapted virus, which is fully competent for replication in this cell type. We compared the sequences of wild-type EIAV and a full-length infectious proviral clone of the cell culture-adapted EIAV and found that the genomes were relatively well conserved with the exception of the envelope gene region, which showed extensive sequence differences. We therefore constructed several wild-type and cell culture-adapted virus chimeras to examine the role of the envelope gene in replication in different cell types in vitro. Unlike wild-type virus, which is restricted by an early event(s) for replication in equine dermis cells, the wild-type outer envelope gene chimeras are replication competent in this cell type. We conclude that even though there are extensive sequence differences between wild-type and cell culture-adapted viruses in the surface envelope gene region, this domain is not a determinant of the differing in vitro cell tropisms.

Immortalization of virus-free human placental cells that express tissue-specific functions.

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.

Cerebellar Purkinje cells provide target support over a limited spatial range: evidence from lurcher chimeric mice

The distribution of Purkinje cells, granule cells, and olivary neurons was quantitatively analyzed in a lurcher +/Lc ↔ C3H/HeJ chimera in which the surviving wild type Purkinje cells were unilaterally distributed in the left hemicerebella. The left hemisphere of this mouse contains 7600 Purkinje cells, approximately 10% of the number of Purkinje cells in inbred C3H/HeJ mice. The right hemisphere contains 300 Purkinje cells, all of which are found within 200 μm of the midline. As in other +/Lc ↔ wild type chimeras, the ratio of granule cells to Purkinje cells is increased in the left hemisphere, reflecting increased granule cell survival. In the right hemisphere, however, the number of granule cells is reduced to that found in +/Lc mutants. In the inferior olive, almost twice as many neurons are found in the right nucleus as opposed to the left nucleus. As the projections of olivary neurons are crossed, the number of olivary neurons is increased in the nuclei that project to the cerebellar hemisphere containing Purkinje cells compared to the olivary nuclei that project to the cerebellar hemisphere with almost no Purkinje cells. The preferential survival of granule cells and olivary neurons that either occupy or project to the hemicerebellum containing Purkinje cells suggests that the availability of trophic support from target Purkinje cell neurons is spatially restricted.

Studies of the dendritic tree of wild‐type cerebellar Purkinje cells in lurcher chimeric mice

Naturally occurring mutations are valuable tools for the study of neural development, especially when used in conjunction with the techniques of chimeric mouse production. In this study we examine the response of Purkinje cell dendrites to the altered developmental environment found in the lurcher in equilibrium with wild-type chimera. Lurcher (+/Lc) is an autosomal dominant gene that causes the cell-autonomous degeneration of all Purkinje cells of +/Lc genotype. Thus, in +/Lc in equilibrium with +/+ chimeras, only wild-type Purkinje cells survive to maturity. The number of these survivors can vary from less than 10,000 to greater than 100,000. Previous work has shown that the final ratio of presynaptic granule cells to postsynaptic Purkinje cells is increased in lurcher chimeras. On average, therefore, one might expect that each remaining Purkinje cell would experience an increased supply of afferents, and our hypothesis was that dendritic growth and/or sprouting might occur as a result. This proved incorrect and, indeed, the Purkinje cells in the lurcher chimeras show changes of a predominantly atrophic nature. Unusual morphologies are found, including variable branching density, failure of the distal dendrite to reach the pial surface, loss of isoplanarity, and the frequent appearance of large caliber, primary or secondary dendritic branches ending abruptly in "stub ends." Quantitative analysis of Golgi-Cox impregnated material reveals that in lurcher chimeras the Purkinje cell dendritic arbor is reduced by more than 60% compared to wild-type animals. We present possible explanations for this finding and consider several potential implications.

Cell lineage dependent and independent control of Purkinje cell number in the mammalian CNS: Further quantitative studies of lurcher chimeric mice

Recent quantitative studies of lurcher chimeric mice have shown that the adult population of cerebellar Purkinje cells can properly be described as a small number of developmental clones of cells. The clones are not seen as patches of contiguous neurons; rather, the cells of any one clone distribute throughout the half-cerebellum that contains them, intermingling extensively with the Purkinje cells of other linkages. Lurcher ⇌ wild-type chimeras were analyzed using the cell autonomous Purkinje-cell-lethal mutant, lurcher ( + L ), as a cell marker. Cell counts from these chimeras revealed that the number of surviving Purkinje cells was always an integral multiple of a unit clone size. These numerical quanta are the evidence for the existence of Purkinje cell developmental clones. When two different inbred strains of mouse were compared (C3H/HeJ and C57BL/6), the resulting clonal analysis showed that the unit clone size (i.e., the number of Purkinje cells in one quantum) is an autonomous property of the lineage and hence, presumably, intrinsic to the progenitor cell that founded it. The current study uses the lurcher chimeric mouse system to examine the cell lineage relationships among the Purkinje cells of a third inbred strain of mouse, AKR/J. The data both support and extend our previous studies. Quantitative analysis reveals that the Purkinje cells of this strain also exist in clones, and the size of these clones is also strain-specific. The number of cells in a single clone (7850), however, is different from either C3H/HeJ (10,200) or C57BL/6 (9200). The fact that this value is so highly polymorphic among the inbred strains of mouse makes it likely that, rather than being a function of different alleles at a single genetic locus, clone size may well represent a multifactorial (but still cell-autonomous) property of developing Purkinje cells. Additional results from a single chimeric animal suggest strongly that clone number (i.e., the number of progenitors selected to found the population) is not strain-specific but results instead from cell:cell interactions during early central nervous system formation.

Role of staggerer gene in determining cell number in cerebellar cortex. II. Granule cell death and persistence of the external granule cell layer in young mouse chimeras

Staggerer mice ( sg/sg) have a severe ataxia correlated with cerebellar Purkinje cell anomalies and granule cell death. Previous studies of adult staggerer ⇹ wild-type chimeric mice have revealed that the mutant Purkinje cell defects visible in the light microscope (small size, ectopia and reduced number) are intrinsic properties of sg/sg Purkinje cells, while the granule cell death observed in the mutant is not an intrinsic property of sg/sg granule cells. In this report we continue the study of granule cell death and examine the phenotype, observed in staggerer mutants, of the developmental persistence of the external granular layer (EGL). Four staggerer ⇹ wild-type mouse chimeras were examined at 16 or 17 days of postnatal age. Most showed intermediate amounts of granule cell pycnosis and all showed intermediate expression of EGL persistence. There was little correlation observed between the amount of granule cell pycnosis and either the genotype of the immediately underlying Purkinje cells, or the proportion of staggerer cells in the chimera as a whole (determined by the genotype ratio of other brain regions and by coat melanocytes). While the presence or absence of an EGL did not correlate with the genotype of the immediately adjacent Purkinje cells, there was good agreement between the extent of persistence of the EGL and the overall contribution of staggerer cells to the chimera. The results suggest that the intermediate numbers of granule cells observed in adult staggerer chimeras are the result of both reduced granule cell genesis and increased pruning by cell death. The findings are further discussed in terms of two possible models of staggerer gene action.

Staggerer chimeras: Intrinsic nature of purkinje cell defects and implications for normal cerebellar development

The site of gene action of the Staggerer mutation of mice was investigated with Staggerer↔wild-type chimeras. Homozygous Staggerer mice show severe locomotor difficulties due to cerebellar abnormalities which include degeneration of virtually all granule cells and cytological defects in Purkinje cells. Although the locomotor deficits of the mutant were not present in the chimeras, the presence of Staggerer cells affected cerebellar structure. The size and the extent of foliation of the chimeric cerebella were intermediate between wild-type and homozygous Staggerer. A normally proportioned granule cell layer was present. Using β-glucuronidase as an independent determinant of a cell's genotype, it was found that the genotypically Staggerer medium-to-large neurons expressed all of the light microscopic defects observable in these cells in the homozygous mutant. These defects include: (1) smaller size; (2) usually ectopic location; and (3) regional variation in the cytological appearance of the perikaryon. By contrast, all Purkinje cells which were genotypically wild-type appeared normal in size, in location and in their cytological appearance. Their density, however, was much reduced from wild-type. The effects of the Staggerer mutation on the granule, stellate and basket cells could not be directly assessed as the glucuronidase marker is not suitable for use with these cells. The Staggerer gene thus acts directly on Purkinje cells rather than via extracellular environmental changes. The findings are discussed in terms of their implications for normal cerebellar development.

Hypoglossal-Facial Anastomosis

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) tumor suppressor gene, is a negative regulator of Ras signaling. Patients with mutations in NF1 have a strong predisposition for cardiovascular disease, which contributes to their early mortality. Nf1 heterozygous (Nf1+/−) bone marrow to wild type chimeras and mice with heterozygous recombination of Nf1 in myeloid cells recapitulate many of the vascular phenotypes observed in Nf1+/− mutants. Although these results suggest that macrophages play a central role in NF1 vasculopathy, the underlying mechanisms are currently unknown. In the present study, we employed macrophages isolated from either Nf1+/− or Lysm Cre+/Nf1f/f mice to test the hypothesis that loss of Nf1 stimulates macropinocytosis in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated that loss of Nf1 in macrophages stimulates macropinocytosis. We next utilized various cellular and molecular approaches, pharmacological inhibitors and genetically modified mice to identify the signaling mechanisms mediating macropinocytosis in Nf1-deficient macrophages. Our results indicate that loss of Nf1 stimulates PKCδ-mediated p47phox phosphorylation via RAS activation, leading to increased NADPH oxidase 2 activity, reactive oxygen species generation, membrane ruffling and macropinocytosis. Interestingly, we also found that Nf1-deficient macrophages internalize exosomes derived from angiotensin II-treated endothelial cells via macropinocytosis in vitro and in the peritoneal cavity in vivo. As a result of exosome internalization, Nf1-deficient macrophages polarized toward an inflammatory M1 phenotype and secreted increased levels of proinflammatory cytokines compared to controls. In conclusion, the findings of the present study demonstrate that loss of Nf1 stimulates paracrine endothelial to myeloid cell communication via macropinocytosis, leading to proinflammatory changes in recipient macrophages.