A variable presentation of Joubert syndrome: Case report and a brief review.

Joubert syndrome is a rare neurological manifestation usually present in late infancy or early childhood with characteristic episodes of abnormal breathing pattern along with the neurological and other systemic involvement.We report a case of confirmed Joubert syndrome present in the immediate neonatal period with isolated spells of oxygen desaturations not accompanied by the classically described breathing pattern and absent neurological symptoms causing delay in the diagnosis. Isolated oxygen desaturation episodes could be a presenting manifestation of Joubert syndrome in a neonatal period.

Successful allogeneic hematopoietic stem cell transplantation for acute myelogenous leukemia in a patient with hepatic echinococcal cyst managed by delayed hepatectomy.

We report the case of a patient who underwent cytotoxic chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) for acute myelogenous leukemia in the presence of hepatic cystic echinococcal infection. The presence of Echinococcus granulosus infection in an immunocompromised host is extremely rare, with lack of established data regarding optimal management. Successful management of the patient's disease processes required a multidisciplinary approach, which included systemic chemotherapy, HSCT, treatment of chronic graft-versus-host-disease, and elective en bloc resection of the hepatic cyst.

Small-cell carcinoma of the urinary bladder. A clinico-pathological study of ten cases.

Small-cell carcinoma (SCC) of the urinary bladder is an infrequent neoplasia accounting for 0.5% of all tumors located at this level. There is a predilection for males over females with a 4:1 proportion and a median age of 66 years. In most cases, the initial diagnosis is made at the metastatic or progressive stage of the disease. For this study, we collected ten cases of SCC of the urinary bladder, diagnosed over a period of 16 years, to describe the morphological and immunocytochemical characteristics of these infrequent neoplasia. In all cases, clinical data such as age at presentation, personal background, clinical symptoms, stage, treatment, clinical outcome and present status were available. Primary antibodies included chromogranin, neuron-specific enolase, synaptophysin, PGP 9.5, HNK-1, cytokeratin 34betaE12, cytokeratin 20, thyroid transcription factor-1 (TTF-1), c-erbB2 (CB-11), p53 (DO7), and Ki67 (MIB-1). In addition to the expression of neural/neuroendocrine markers, immunostaining for p53 and c-erbB2 was found in 80% and 50% of cases, respectively. In this paper, we confirm the aggressive course of the neoplastic disease. The expression of c-erbB2 in 50% of cases opens up hypothetical new possibilities for the use of immunotherapy in such cases.

Malignant granular cell tumor of soft tissues: a study of two new cases.

We describe 2 cases of malignant granular cell tumor. A marked discrepancy exists concerning the criteria of malignancy of this rare entity, which was diagnosed in male patients aged 41 and 52 years, respectively. They presented with tumors measuring more then 3.5 cm; one arose in the dermis and subcutaneous tissue in the region of the scapula, whereas the other was situated in deeper soft tissue in the pelvis. One case, with previous diagnosis of benign granular cell tumor, presented local recurrence 2 years after the initial diagnosis. The other case presented a fascicular pattern of growth invading adjacent muscular tissue. Both neoplasms were composed of polygonal and spindle cells, showing abundant cytoplasm, vesicular nuclei with large nucleoli, high nuclear-to-cytoplasmic ratio, and pleomorphism. Immunohistochemically, the neoplastic cells of both cases were positive for vimentin, S100 protein, neuron-specific enolase (NSE), and CD68. In addition, high expression of p53 and MiB1 (Ki67) was detected. Herein, we discuss the morphologic and immunohistochemical criteria of malignancy of granular cell tumors. Int J Surg Pathol 9(3):255-259, 2001

PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor.

Platelet-derived growth factors (PDGFs) are important in many types of mesenchymal cell. Here we identify a new PDGF, PDGF-C, which binds to and activates the PDGF alpha-receptor. PDGF-C is activated by proteolysis and induces proliferation of fibroblasts when overexpressed in transgenic mice. In situ hybridization analysis in the murine embryonic kidney shows preferential expression of PDGF-C messenger RNA in the metanephric mesenchyme during epithelial conversion. Analysis of kidneys lacking the PDGF alpha-receptor shows selective loss of mesenchymal cells adjacent to sites of expression of PDGF-C mRNA; this is not found in kidneys from animals lacking PDGF-A or both PDGF-A and PDGF-B, indicating that PDGF-C may have a unique function.

Fate of the mammalian cardiac neural crest.

A subpopulation of neural crest termed the cardiac neural crest is required in avian embryos to initiate reorganization of the outflow tract of the developing cardiovascular system. In mammalian embryos, it has not been previously experimentally possible to study the long-term fate of this population, although there is strong inference that a similar population exists and is perturbed in a number of genetic and teratogenic contexts. We have employed a two-component genetic system based on Cre/lox recombination to label indelibly the entire mouse neural crest population at the time of its formation, and to detect it at any time thereafter. Labeled cells are detected throughout gestation and in postnatal stages in major tissues that are known or predicted to be derived from neural crest. Labeling is highly specific and highly efficient. In the region of the heart, neural-crest-derived cells surround the pharyngeal arch arteries from the time of their formation and undergo an altered distribution coincident with the reorganization of these vessels. Labeled cells populate the aorticopulmonary septum and conotruncal cushions prior to and during overt septation of the outflow tract, and surround the thymus and thyroid as these organs form. Neural-crest-derived mesenchymal cells are abundantly distributed in midgestation (E9.5-12.5), and adult derivatives of the third, fourth and sixth pharyngeal arch arteries retain a substantial contribution of labeled cells. However, the population of neural-crest-derived cells that infiltrates the conotruncus and which surrounds the noncardiac pharyngeal organs is either overgrown or selectively eliminated as development proceeds, resulting for these tissues in a modest to marginal contribution in late fetal and postnatal life.

Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice.

Using gene trap mutagenesis, we have identified a mutation in mice that causes exencephaly, acrania, facial clefting, and spina bifida, all of which can be attributed to failed neural tube closure. This mutation is designated shroom (shrm) because the neural folds "mushroom" outward and do not converge at the dorsal midline. shrm encodes a PDZ domain protein that is involved at several levels in regulating aspects of cytoarchitecture. First, endogenous Shrm localizes to adherens junctions and the cytoskeleton. Second, ectopically expressed Shrm alters the subcellular distribution of F-actin. Third, Shrm directly binds F-actin. Finally, cytoskeletal polarity within the neuroepithelium is perturbed in mutant embryos. In concert, these observations suggest that Shrm is a critical determinant of the cellular architecture required for proper neurulation.

Compartmentalized signaling by GPI-anchored ephrin-A5 requires the Fyn tyrosine kinase to regulate cellular adhesion.

Eph receptor tyrosine kinases and their corresponding surface-bound ligands, the ephrins, provide cues to the migration of cells and growth cones during embryonic development. Here we show that ephrin-A5, which is attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidylinositol-anchor, induces compartmentalized signaling within a caveolae-like membrane microdomain when bound to the extracellular domain of its cognate Eph receptor. The physiological response induced by this signaling event is concomitant with a change in the cellular architecture and adhesion of the ephrin-A5-expressing cells and requires the activity of the Fyn protein tyrosine kinase. This study stresses the relevance of bidirectional signaling involving the ephrins and Eph receptors during brain development.

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling.

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

PDGFB regulates the development of the labyrinthine layer of the mouse fetal placenta.

PDGFB is a growth factor which is vital for the completion of normal prenatal development. In this study, we report the phenotypic analysis of placentas from mouse conceptuses that lack a functional PDGFB or PDGFRbeta gene. Placentas of both types of mutant exhibit changes in the labyrinthine layer, including dilated embryonic blood vessels and reduced numbers of both pericytes and trophoblasts. These changes are seen from embryonic day (E) 13.5, which coincides with the upregulation of PDGFB mRNA levels in normal placentas. By E17, modifications in shape, size, and number of the fetal blood vessels in the mutant placentas cause an abnormal ratio of the surface areas between the fetal and the maternal blood vessels in the labyrinthine layer. Our data suggest that PDGFB acts locally to contribute to the development of the labyrinthine layer of the fetal placenta and the formation of a proper nutrient-waste exchange system during fetal development. We point out that the roles of PDGFB/Rbeta signaling in the placenta may be analogous to those in the developing kidney, by controlling pericytes in the labyrinthine layer and mesangial cells in the kidney.

Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis.

Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.

Src family kinases are required for integrin but not PDGFR signal transduction.

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors.

EGF receptor signaling stimulates SRC kinase phosphorylation of clathrin, influencing clathrin redistribution and EGF uptake.

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

Growth factor signaling pathways in vascular development.

Recent research on the formation and maintenance of the vasculature in the embryo and in the adult has provided a greater understanding of the cellular signals involved in these processes. With this understanding comes the potential means of controlling vascularization in pathological situations such as tumorigenesis and wounding. For the purpose of this review, we will discuss the key receptor tyrosine kinases involved in vascular function and the molecules which relay signals downstream of receptor activation. The receptor tyrosine kinases discussed include the vascular endothelial cell growth factor receptors, Eph receptors, Tie1, and Tie2, all of which are expressed on vascular endothelial cells. We also discuss the roles of the platelet derived growth factor receptors which are expressed on vascular smooth muscle cells. While all of these receptor tyrosine kinases activate many similar effector molecules, some of the signals initiated appear to be distinct. This may explain, at least in part, how different receptor tyrosine kinases expressed in overlapping patterns on the developing vasculature, direct unique biological functions.

Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli.

Kidney glomerulus mesangial cells fail to develop in mice carrying targeted null mutations in the platelet-derived growth factor (PDGF)-B or PDGF-Rbeta genes. We have examined the pattern of expression of these genes and smooth muscle markers during kidney development, to address the possible mechanisms underlying the mutant phenotypes. In wild-type embryos, PDGF-B was expressed in vascular endothelial cells, particularly in capillary endothelial cells in the developing glomeruli, whereas PDGF-Rbeta was found in perivascular mesenchymal cells in the developing renal cortex. In the course of glomerular development, small groups of PDGF-Rbeta and desmin-expressing cells collected in the 'S'-shaped and early cup-shaped vesicles, and at later stages such cells were found in the glomerular mesangium. In PDGF-B or -Rbeta null embryos, some PDGF-Rbeta/desmin or desmin-positive cells, respectively, were seen in early cup-shaped vesicles, but fewer than in the wild type, and further development of the mesangium failed. In mouse chimeras composed of PDGF-Rbeta +/+ and -/- cells, the Rbeta-/- cells failed to populate the glomerular mesangium. Our results show that while the mesangial cell lineage is specified independently of PDGF-B/Rbeta, these molecules provide critical permissive signals in mesangial cell development. We propose a model in which mesangial cells originate from PDGF-Rbeta-positive progenitors surrounding the developing glomerular afferent and efferent arterioles, and are co-recruited in response to PDGF-B during angiogenic formation of the glomerular capillary tuft.

Chimaeric analysis reveals role of Pdgf receptors in all muscle lineages.

Blood vessels originate as simple endothelial cell tubes. It has been proposed that platelet-derived growth factor B polypeptide (Pdgfb) secreted by these endothelial cells drives the formation of the surrounding muscular wall by recruiting nearby mesenchymal cells. However, targetted inactivation of the Pdgfb gene or the Pdgf receptor beta (Pdgfrb) gene, by homologous recombination, does not prevent the development of apparently normal large arteries and connective tissue. We have used an in vivo competition assay in which we prepared chimaeric blastocysts, composed of a mixture of wild-type (Pdgfrb[+/+]) and Pdgfrb(+/-) or wild-type and Pdgfrb(-/-) cells, and quantified the relative success of cells of the two component genotypes in competing for representation in different cell lineages as the chimaeric embryos developed. This study revealed that the participation of Pdgfrb(-/-) cells in all muscle lineages (smooth, cardiac, skeletal and pericyte) was reduced by eightfold compared with Pdgfrb(+/+) cells, and that participation of Pdgfrb(+/-) cells was reduced by twofold (eightfold for pericytes). Pdgfrb inactivation did not affect cell contribution to non-muscle mesodermal lineages, including fibroblasts and endothelial cells. Chimaera competition is therefore a sensitive, quantitative method for determining developmental roles of specific genes, even when those roles are not apparent from analysis of purebred mutants; most likely because they are masked by homeostatic mechanisms.

Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation.

The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, we disrupted the Mad1 gene by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with a compensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. In addition, recovery of the peripheral granulocyte compartment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation.

Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells.

The ROSA beta geo26 (ROSA26) mouse strain was produced by random retroviral gene trapping in embryonic stem cells. Staining of ROSA26 tissues and fluorescence-activated cell sorter-Gal analysis of hematopoietic cells demonstrates ubiquitous expression of the proviral beta geo reporter gene, and bone marrow transfer experiments illustrate the general utility of this strain for chimera and transplantation studies. The gene trap vector has integrated into a region that produces three transcripts. Two transcripts, lost in ROSA26 homozygous animals, originate from a common promoter and share identical 5' ends, but neither contains a significant ORF. The third transcript, originating from the reverse strand, shares antisense sequences with one of the noncoding transcripts. This third transcript potentially encodes a novel protein of at least 505 amino acids that is conserved in humans and in Caenorhabditis elegans.