bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytologic‐histologic correlation of nongynecologic cytopathology cases: Separation of determinate from indeterminate cytologic diagnoses for analysis and monitoring of laboratory performance
Authors: Ohori N. Paul , Schoedel Karen E. , Rajendiran Swaminathan
Published: January 2003

Much of the literature on the quality‐assurance aspect of cytologic‐histologic correlation (CHC) has focused on gynecologic cytology. For nongynecologic cytopathology, the process is complicated by the use of determinate (positive for malignant cells, negative for malignant cells) and indeterminate (atypical, suspicious, or follicular lesion) diagnostic categories. Here, we illustrate our routine methodology for analyzing CHC data on nongynecologic cytopathology cases by separating determinate from indeterminate cases. A focused list of determinate and indeterminate cytopathology cases with surgical pathology correlation is generated each week. The determinate cases are ascertained as true positive (TP), true negative (TN), false positive (FP), or false negative (TN). The discrepant cases (FP and FN) are investigated to determine the cause (sampling, interpretation, or screening). For indeterminate cases, the surgical pathology outcome (benign, malignant) and suitability of the cytopathology category utilized are reviewed. For the focused period of 4 mo, sensitivity was 70% and specificity was 100%. The most common reason for false‐negative diagnoses was a sampling problem in the cytologic specimen; there were no false‐positive diagnoses. Malignant outcomes for follicular lesion, atypical, and suspicious diagnoses were 29%, 40%, and 76%, respectively. Data derived from regularly performed CHC are useful in reviewing the diagnostic performance of the laboratory. Diagn. Cytopathol. 2003;28:28–34. © 2003 Wiley‐Liss, Inc.
Diagnosis and grading of chondrosarcomas on FNA biopsy material
Authors: Lerma Enrique , Tani Edneia , Brosjö Otte , Bauer Henrik , Söderlund Veli , Skoog Lambert
Published: January 2003

A consecutive series of 47 chondrosarcomas was reviewed to identify the most useful criteria for cytological diagnosis, and grading in comparison to histology grade. This series includes 39 evaluable cases, including 2 patients with Ollier's disease and 2 with extraskeletal chondrosarcomas. Eight cases were excluded because of lack of histologic confirmation (3), nondiagnostic cytology (4), and one inconclusive histologic diagnosis. The cytologic diagnosis of these tumors was based on the identification of chondrocytes and a chondromyxoid stroma. The chondrocytes often showed double nuclei (34/39) and macronucleoli (20/39). Nuclear atypia in terms of distinct variation in size was less common (16/39). Cytoplasmic granules and vacuoles were often seen (34/39 and 29/39, respectively). In smears, chondroid fragments and a chondromyxoid backgroud were seen in a majority of cases (36/39 and 30/39, respectively). By grade, concordance with histology was present in 4/7 grade I tumors, 26/29 grades II–III, and 5/5 grade IV, respectively. Diagn. Cytopathol. 2003;28:13–17. © 2003 Wiley‐Liss, Inc.
Percutaneous image‐guided fine‐needle aspiration of peritoneal lesions
Authors: Layfield Lester J. , Gopez Evelyn V.
Published: January 2003

Fine‐needle aspiration (FNA) is a widely accepted technique for the initial tissue diagnosis of a variety of lesions arising within retroperitoneal and intraabdominal viscera. Fear of complications secondary to perforation of the bowel wall has limited the use of FNA in the diagnosis of gastrointestinal and peritoneal masses. A variety of primary and secondary neoplasms involving the peritoneum may present as multiple nodules, as masses, or as diffuse involvement of the peritoneum. When these lesions are associated with mass lesions or areas of significant peritoneal thickening, they become amenable to percutaneous image‐guided FNA. We report on our experience with a series of 23 peritoneal lesions investigated by FNA for which subsequent histologic confirmation was available in 19, along with an additional 4 cases without histologic confirmation. One to four passes were made into each lesion, and immediate assessment for adequacy was performed by a cytopathologist in all cases. All 17 cases with a specific cytologic diagnosis and histologic confirmation represented either primary or metastatic neoplasms (5 gastrointestinal stromal tumors, 4 metastatic melanomas, 2 mesotheliomas, 1 lymphoma, 1 example of Kaposi's sarcoma, 1 serous papillary carcinoma of ovarian origin, 1 mucinous adenocarcinoma of ovarian origin, 1 intraabdominal desmoplastic small‐cell tumor, and 1 solitary fibrous tumor of the peritoneum). In an additional 4 cases, the aspirates were judged as insufficient for diagnosis, with the smears containing only blood and benign mesothelial cells and/or inflammatory cell elements. These four smears were associated with both neoplastic and nonneoplastic lesions. Surgical confirmation was obtained in only 2 of these cases (1 metastatic melanoma and 1 example of omental and peritoneal involvement by an ovarian adenocarcinoma). Two cases without histologic confirmation were associated with clinically confirmed metastases. In our series, no acute or chronic postprocedural complications were identified, indicating that FNA in this setting is a safe technique. Accurate cytologic diagnosis was achieved in 74% of cases. The overall insufficiency rate was 26%. Diagn. Cytopathol. 2003;28:6–12. © 2003 Wiley‐Liss, Inc.
Immunocytochemistry of serous effusion specimens: A comparison of ThinPrep® vs. cell block
Authors: Gong Yun , Sun Xiaoping , Michael Claire W. , Attal Sam , Williamson Beth A. , Bedrossian Carlos W.M.
Published: January 2003

The ThinPrep® Processor is gaining popularity in both gynecologic and nongynecologic cytologic samples, including effusion specimens. We compared immunocytochemical results on ThinPrep and cell‐block preparations from the same effusion specimen with antibodies commonly used in effusion cytopathology. Samples from 17 reactive effusions and 79 effusions with metastatic adenocarcinomas were each prepared as monolayer ThinPreps and formalin‐fixed, paraffin‐embedded cell blocks. All slides were immunostained with antibodies against intermediate filaments (cytokeratins 8 and 18, and vimentin), cytoplasmic or membrane proteins (EMA, polyclonal CEA, B72.3, and BerEP4), and nuclear antigens (Ki67, PCNA, and p53), using the avidin‐biotin procedure. ThinPrep and cell‐block slides were stained simultaneously, using identical protocols. Both preparations showed similar results with respect to frequency of positivity, intensity, and distribution of stain for all nonnuclear markers tested. However, for the three nuclear markers Ki67, PCNA, and p53, the frequency and intensity of reaction with ThinPrep were significantly lower than with the cell‐block preparation, particularly in malignant effusions. These findings suggest that immunocytochemical results obtained with ThinPrep® match those of cell block for most markers tested. However, cell‐block preparation are superior to ThinPrep for nuclear markers (Ki67, PCNA, and p53). Diagn. Cytopathol. 2003;28:1–5. © 2003 Wiley‐Liss, Inc.
False‐positive squamous cell carcinoma in cervical smears: Cytologic‐histologic correlation in 19 cases
Authors: Levine Pascale Hummel , Elgert Paul A. , Mittal Khush
Published: January 2003

Cytologic features of squamous intraepithelial lesions (SIL) can mimic those of invasive squamous‐cell carcinoma. We compare and correlate the cytological findings of 19 false‐positive squamous‐cell carcinomas with follow‐up cone biopsies or hysterectomy specimens to define which type of dysplasia is more prone to diagnostic errors on cervical Papanicolaou (Pap) smears. Out of 128 patients diagnosed with invasive squamous‐cell carcinoma from 1994–2000, 19 (14.8%) with follow‐up cone biopsies or hysterectomy specimens were false‐positive cases, showing only cervical intraepithelial neoplasia (CIN). We reviewed tissue sections from these 19 cases of CIN for cytologic features of squamous‐cell carcinoma, such as markedly pleomorphic and/or dysplastic squamous cells, necrosis, and nucleoli. Twelve of 19 patients (63%) were menopausal. The mean age was 50.5 yr. On review of cervical smears, 18 cases qualified for the cytologic diagnosis of squamous‐cell carcinoma, keratinizing type, and one case qualified for squamous‐cell carcinoma, nonkeratinizing type. Pleomorphic and/or keratinizing dysplasia was found in 15 out of 19 patients (79%), necrosis within superficial endocervical glands in 9 out of 19 patients (47%), and conspicuous nucleoli in 12 out of 19 patients (63%). One or more of these changes were seen in all but 2 patients (89%). Endocervical gland involvement was present and extensive in 18 of the 19 cases (94%). The mean age was older than expected for SIL (50.5 vs. a reported 40), and matched the mean age found in patients with invasive squamous‐cell carcinoma. Pleomorphic and/or keratinizing dysplasia involving endocervical glands may exhibit the cytologic features of squamous‐cell carcinoma on cervical Pap smears. Diagn. Cytopathol. 2003;28:23–27. © 2003 Wiley‐Liss, Inc.
Quick aspiration cytology for thyroid nodules by modified Ultrafast Papanicolaou staining
Authors: Maruta Junko , Hashimoto Hironobu , Yamashita Hiroto , Yamashita Hiroyuki , Noguchi Shiro
Published: January 2003

Quick cytologic diagnosis by Ultrafast Papanicolaou stain (Ultrafast stain) is useful in various neoplastic diseases; however, it has a significant drawback in that the ground‐glass appearance of nuclei is a preparation artifact in some specimens from thyroid aspirations. To eliminate this artifact, the nucleus was stained with Gill‐5 Hematoxylin (modified Ultrafast stain) rather than with Richard‐Allan Hematoxylin 2 in Ultrafast stain. The applicability of this modified Ultrafast stain for quick diagnosis of thyroid diseases was investigated. Two specimens from each of 251 thyroid aspirations (122 malignant and 131 benign) were prepared using the modified Ultrafast stain and the standard Papanicolaou stain. The sensitivities of cytologic diagnosis in specimens stained by the standard Papanicolaou method and the modified Ultrafast method were 95.0% and 93.3%, respectively, and the specificities were 99.2% and 97.7%, respectively. The modified Ultrafast stain is a reliable procedure for quick diagnosis in thyroid aspiration cytology. Diagn. Cytopathol. 2003;28:45–48. © 2003 Wiley‐Liss, Inc.
Genetic linkage maps of the West African clawed frog Xenopus tropicalis
Authors: Kochan Kelli J. , Wright David A. , Schroeder Lisa J. , Shen Jianjun , Morizot Donald C.
Published: January 2003

Amphibians, and particularly the African clawed frog Xenopus laevis, have been used for more than a century as models of vertebrate embryonic development. However, in many cases, elucidation of developmental functions of specific gene sequences could be severely impeded, because X. laevis is a tetraploid species, with multiple functional copies of many genes of interest. Recent studies have shifted focus to the West African or tropical clawed frog, X. tropicalis, the only known diploid species of the genus Xenopus. Here, we present two preliminary linkage maps, constructed by analysis of joint segregation of amplified fragment length polymorphism (AFLP) markers in a X. tropicalis interstrain hybrid. A total of 53 markers, including 51 AFLP markers and 2 isozyme markers, are presently assigned to 13 multipoint linkage groups on a map of the maternal strain, whereas 9 AFLP markers from the paternal strain are assigned to 3 linkage groups on a separate map. A dense genetic linkage map is essential in mapping new developmental mutants and determining their sequences by positional cloning. © 2002 Wiley‐Liss, Inc.
Genome‐wide examination of myoblast cell cycle withdrawal during differentiation
Authors: Shen Xun , Collier J. Michael , Hlaing Myint , Zhang Leanne , Delshad Elizabeth H. , Bristow James , Bernstein Harold S.
Published: January 2003

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up‐regulation of MyoD, followed by myogenin, and activation of Bcl‐2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21WAF1/Cip1 increased and α‐actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome‐wide screen to identify other gene products involved in this process. Using microarrays containing ∼10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT‐PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly‐6A, whose expression specifically was up‐regulated during cell cycle withdrawal coincident with early myoblast differentiation. 2002 Wiley‐Liss, Inc.
Molecular components of the endoderm specification pathway in Xenopus tropicalis
Authors: D'Souza Anjali , Lee Monica , Taverner Nicola , Mason Julia , Carruthers Samantha , Smith James C. , Amaya Enrique , Papalopulu Nancy , Zorn Aaron M.
Published: January 2003

Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate endoderm specification. However, loss‐of‐function analysis is required to resolve the precise function of the genes involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17α, tSox17β, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes, thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better probe penetration than in laevis whole‐mount in situ hybridizations—allowing us to visualize transcripts in the deep endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large number of tropicalis‐expressed sequence tags that are now available. © 2002 Wiley‐Liss, Inc.
Spatial and temporal expression of folate‐binding protein 1 (Fbp1) is closely associated with anterior neural tube closure in mice
Authors: Saitsu Hirotomo , Ishibashi Makoto , Nakano Hitoo , Shiota Kohei
Published: January 2003

Periconceptional folate supplementation is widely believed to have significant preventive effects on the production of neural tube defects. Folate‐binding protein 1 (FBP1) is one of the membrane proteins that mediate cellular uptake of folate. Although recent studies suggest that Fbp1 is essential for neural tube closure, the pattern of Fbp1 expression in embryonic tissues has not been examined in detail. To elucidate how Fbp1 contributes to neural tube closure, we examined the spatial and temporal expression patterns of Fbp1 in the developing neural folds and tube of mouse embryos by in situ hybridization. Fbp1 showed a distinct expression pattern in the neural folds, which preceded initiation of neural tube closure at the cervical region and the prosencephalic/mesencephalic boundary. Fbp1 expression was mainly localized to the most dorsal regions of the neural folds where fusion was to occur. With proceeding of neural fold fusion, Fbp1 expression extended to the adjacent unfused neural folds. In the rhombencephalon, robust expression of Fbp1 was observed in rhombomere2 (r2) and r6, suggesting its roles in development of neural crest cells. Fbp1 also showed intense expression in the yolk sac, indicating that FBP1 may mediate transferring maternal folate to embryos during neurulation. These findings indicate close association between Fbp1 and anterior neural tube closure. © 2002 Wiley‐Liss, Inc.
In ovo electroporation of Crim1 in the developing chick spinal cord
Authors: Kolle Gabriel , Jansen Annemieke , Yamada Toshiya , Little Melissa
Published: January 2003

The novel mammalian gene Crim1 encodes a transmembrane bound protein with similarity to the secreted bone morphogenetic protein (BMP) antagonists, vertebrate Chordin, and its Drosophila homologue short gastrulation. Crim1 is expressed in the neural tube in mouse in a restricted pattern, but its function in central nervous system development is largely unknown. We isolated the chicken Crim1 orthologue and analyzed its expression in the developing neural tube. Chicken CRIM1 shares strong homology to human/mouse CRIM1 and C. elegans CRIM1‐like proteins. Crim1 is expressed in a similar but not identical pattern to that in the developing spinal cord of mouse, including the notochord, floor plate, motor neurons, and the roof plate. Unlike follistatin, a secreted inhibitor of BMPs, in ovo electroporation of CRIM1, as a full‐length transmembrane bound or secreted ectodomain was not sufficient to disrupt early patterning of the neural tube. However, ectodomain CRIM1 overexpression leads to an approximate 50% decrease in populations of specific ventral neuronal populations, including ISL‐1+ motor neurons, CHX‐10+ V1, and EN‐1+ V2 interneurons. © 2002 Wiley‐Liss, Inc.
Pronephric duct extension in amphibian embryos: Migration and other mechanisms
Authors: Drawbridge Julie , Meighan Christopher M. , Lumpkins Rebecca , Kite Mary E.
Published: January 2003

Initiation of excretory system development in all vertebrates requires (1) delamination of the pronephric and pronephric duct rudiments from intermediate mesoderm at the ventral border of anterior somites, and (2) extension of the pronephric duct to the cloaca. Pronephric duct extension is the central event in nephric system development; the pronephric duct differentiates into the tubule that carries nephric filtrate out of the body and induces terminal differentiation of adult kidneys. Early studies concluded that pronephric ducts formed by means of in situ segregation of pronephric duct tissue from lateral mesoderm ventral to the forming somites; more recent studies highlight caudal migration of the pronephric duct as the major morphogenetic mechanism. The purpose of this review is to provide the historical background on studies of the mechanisms of amphibian pronephric duct extension, to review evidence showing that different amphibians perform pronephric duct morphogenesis in different ways, and to suggest future studies that may help illuminate the molecular basis of the mechanisms that have evolved in amphibians to extend the pronephric duct to the cloaca. © 2002 Wiley‐Liss, Inc.
T‐box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lung
Authors: Cebra‐Thomas Judith A. , Bromer Jason , Gardner Rose , Lam Gordon K. , Sheipe Hillary , Gilbert Scott F.
Published: January 2003

The regulation of signaling pathways is a prerequisite for coordinating the induction between mesenchymal and epithelial tissues during morphogenesis. Mesenchymal FGF10 is known to be an important paracrine factor regulating the branching morphogenesis of the bronchial epithelium. By using antisense oligonucleotides (AS ODNs) and in vitro culture of embryonic lungs, we demonstrate that the transcription factors Tbx4 and Tbx5 are critical for the expression of mesenchymal FGF10. Treatment of embryonic lung cultures with AS ODNs to Tbx4 and Tbx5 reduces the level of these transcripts, suppresses Fgf10 expression in the mesenchyme, and completely eliminates the formation of new lung branches. If FGF10 is locally replaced in these AS ODN‐treated lungs, epithelial branching is restored. These studies provide evidence that the production of branching signals by the lung mesenchyme is mediated by T‐box genes. © 2002 Wiley‐Liss, Inc.
Drosophila NAB (dNAB) is an orphan transcriptional co‐repressor required for correct CNS and eye development
Authors: Clements Mark , Duncan Dianne , Milbrandt Jeffrey
Published: January 2003

The mammalian NAB proteins have been identified previously as potent co‐repressors of the EGR family of zinc finger transcription factors. Drosophila NAB (dNAB), like its mammalian counterparts, binds EGR1 and represses EGR1‐mediated transcriptional activation from a synthetic promoter. In contrast, dNAB does not bind the Drosophila EGR‐related protein klumpfuss. dnab RNA is expressed exclusively in a subset of neuroblasts in the embryonic and larval central nervous system (CNS), as well as in several larval imaginal disc tissues. Here, we describe the creation of targeted deletion mutations in the dnab gene and the identification of additional, EMS‐induced dnab mutations by genetic complementation analysis. Null alleles in dnab cause larval locomotion defects and early larval lethality (L1–L2). A putative hypomorphic allele in dnab instead causes early adult lethality due to severe locomotion defects. In the dnab ‐/‐ CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that dnab ‐/‐ clones are either very small or absent. Similarly, dNAB overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye‐specific phenotypes will prove useful for enhancer/suppressor screens to identify dnab‐interacting genes. © 2002 Wiley‐Liss, Inc.
Cyclops‐independent floor plate differentiation in zebrafish embryos
Authors: Albert Stéphanie , Müller Ferenc , Fischer Nadine , Biellmann Dominique , Neumann Carl , Blader Patrick , Strähle Uwe
Published: January 2003

In zebrafish, development of the ventral neural tube depends on the Nodal‐related signal Cyclops (Cyc). One‐day‐old cyc mutant embryos lack the medial floor plate (MFP). We show here that cells expressing MFP marker genes differentiate gradually in cyc mutant embryos in a delayed manner during the second day of development. This late differentiation is restricted to the hindbrain and spinal cord and depends on an intact Hedgehog (Hh) signalling pathway. Cells expressing MFP marker genes in cyc mutant embryos appear to be derived from lateral floor plate (LFP) cells as they coexpress LFP and MFP marker genes. This finding suggests that the correct temporal development of the MFP is required for the distinction of LFP and MFP cells in wild‐type embryos. © 2002 Wiley‐Liss, Inc.
Rho‐associated kinases play an essential role in cardiac morphogenesis and cardiomyocyte proliferation
Authors: Zhao Zhiyong , Rivkees Scott A.
Published: January 2003

Rho‐associated coiled‐coil kinases (ROCKs), initially identified as effectors for Rho GTPases, play a role in cardiac cell physiology and are also expressed in the developing heart. However, their role in cardiac development is not known. To investigate the role of these kinases in cardiac development, we examined cardiac development in cultured murine embryos treated with the ROCK inhibitor Y27632. After inhibition of ROCK activity, we found disturbed cardiac chamber formation and trabeculation. To further examine the mechanisms by which ROCK blockade causes cardiac hypoplasia, we assessed programmed cell death and cell proliferation in the hearts. We found decreased cell proliferation in the Y27632‐treated hearts, but no changes in programmed cell death. We further observed that ROCK inhibition decreased cardiac myocyte proliferation, suggesting that ROCK kinases regulate cardiomyocyte division. To identify factors involved in ROCK action in regulation of cardiac cell division, we examined expression of cell cycle proteins by using Western blot analysis. We found that ROCK blockade decreased expression of cell cycle proteins, cyclin D3, CDK6, and p27KIP1 in the hearts and cardiomyocytes, which are required for initiation of cell cycle and G1/S phase transition. These observations show that ROCK kinases play a role in cardiac development and that ROCK kinases regulate cardiac cell proliferation and cell cycle protein expression. © 2002 Wiley‐Liss, Inc.
Heart‐selective expression of the chicken FK506‐binding protein (FKBP) 12.6 gene during embryonic development
Authors: Yazawa Shigenobu , Obata Koji , Iio Akio , Koide Masafumi , Yokota Mitsuhiro , Sasaki Shin‐Ichi , Kagami Hiroshi , Ono Tamao
Published: January 2003

FKBP12.6, a member of the family of FK506‐binding proteins, selectively associates with the cardiac isoform of the ryanodine receptor and thereby stabilizes this Ca2+ release channel. A chicken FKBP12.6 (chFKBP12.6) cDNA was cloned and shown to encode a protein of 108 amino acids. The deduced amino acid sequence of chFKBP12.6 is 91–92% identical to those of mammalian FKBP12.6 proteins. Northern blot analysis revealed that chFKBP12.6 mRNA is largely restricted to the heart during embryonic development and that the abundance of this mRNA in the heart decreases, and it becomes restricted to the atrium during cardiogenesis. In situ hybridization revealed that chFKBP12.6 mRNA is localized to the precardiac mesoderm before formation of the primitive heart tube. Expression of the chFKBP12.6 gene was initially apparent throughout the developing multichambered heart but became restricted to the atria before hatching. Reverse transcription and polymerase chain reaction analysis demonstrated that chFKBP12.6 mRNA is present in the embryo from early gastrulation and is most abundant immediately after the onset of the heartbeat. These observations suggest that the chFKBP12.6 gene is expressed before heart morphogenesis to play a role in excitation–contraction coupling in cardiomyocytes and that the function of the encoded protein becomes increasingly restricted to the atrium during embryonic development. © 2002 Wiley‐Liss, Inc.
Developmental expression of the HNK‐1 carbohydrate epitope on aggrecan during chondrogenesis
Authors: Domowicz Miriam S. , Mueller Melissa M. , Novak Todd E. , Schwartz Lauren E. , Schwartz Nancy B.
Published: January 2003

Previously, we showed that the HNK‐1 carbohydrate epitope is expressed on aggrecan synthesized in the notochord but not in mature cartilage. In the present study, we demonstrate that in immature cartilage (embryonic day 6) the HNK‐1 epitope is also expressed predominantly on aggrecan proteoglycan molecules. This finding was verified by using an aggrecan‐deficient mutant, the nanomelic chick, which lacks HNK‐1 immunostaining in the extracellular matrix of dividing and hypertrophic chondrocytes as late as embryonic day 12. By using both biochemical and immunologic approaches, the initially prominent expression of the HNK‐1 epitope is down‐regulated as development of limb and vertebral cartilage proceeds, so that by embryonic day 14 no HNK‐1 is detectable. Localization changes with development and the HNK‐1–aggrecan matrix becomes restricted to dividing and hypertrophic chondrocytes and is particularly concentrated in the intraterritorial matrix. Concomitant with the temporal and spatial decreases in HNK‐1, there is a significant increase in keratan‐sulfate content and the aggrecan‐borne HNK‐1 epitope is closely associated with proteolytic peptides that contain keratan sulfate chains, rather than chondroitin sulfate chains or carbohydrate‐free domains. Lastly, the diminution in HNK‐1 expression is consistent with a reduction in mRNA transcripts specific for at least one of the key enzymes in HNK‐1 oligosaccharide biosynthesis, the HNK‐1 sulfotransferase. These findings indicate that the HNK‐1 carbohydrate may be a common modifier of several proteoglycans (such as aggrecan) that are usually expressed early in development, and that HNK‐1 addition to these molecules may be regulated by tissue‐ and temporal‐specific expression of requisite sulfotransferases and glycosyltransferases. © 2002 Wiley‐Liss, Inc.
T‐box binding site mediates the dorsal activation of myf‐5 in Xenopus gastrula embryos
Authors: Lin Gu Fa , Geng Xin , Chen Ying , Qu Bin , Wang Fubin , Hu Ruiying , Ding Xiaoyan
Published: January 2003

Myf‐5, a member of the muscle regulatory factor family of transcription factors, plays an important role in the determination, development, and differentiation of the skeletal muscle. Factors that regulate the expression of myf‐5 itself are not well understood. We show here that a T‐box binding site in the Xenopus myf‐5 promoter mediated the activation of myf‐5 expression through specific interaction with nuclear proteins of gastrula embryos. The T‐box binding site could be bound by and respond to T‐box proteins. T‐box genes could induce Xmyf‐5. The results suggest that T‐box proteins are involved in the specification of myogenic mesoderm and muscle development. © 2002 Wiley‐Liss, Inc.
Ci‐GATAa, a GATA‐class gene from the ascidian Ciona intestinalis: Isolation and developmental expression
Authors: D'Ambrosio Palmira , Fanelli Albertina , Pischetola Michele , Spagnuolo Antonietta
Published: January 2003

Members of the GATA family of zinc finger transcription factors have been shown to play important roles in controlling gene expression in a variety of cell types in many metazoan. Here, we describe the identification of Ci‐GATAa, a member of this gene family, in the ascidian Ciona intestinalis. Whole‐mount in situ hybridization showed that Ci‐GATAa was expressed in a highly dynamic manner. The maternal transcript was evenly distributed in the embryo during early stages of development; however, the signal gradually decreased until it disappeared at the 64‐cell stage. A zygotic transcript was detected at the 110‐cell stage in the blastomeres precursors of three different tissues (brain vesicle, mesenchyme, and trunk lateral cells) and the signal was conserved in these territories up to the larval stage, indicating an important role for Ci‐GATAa during ascidian differentiation. © 2002 Wiley‐Liss, Inc.
Chick limbs with mouse teeth: An effective in vivo culture system for tooth germ development and analysis
Authors: Koyama Eiki , Wu Changshan , Shimo Tsuyoshi , Pacifici Maurizio
Published: January 2003

Mouse tooth germ development is currently studied by three main approaches: in wild‐type and mutant mouse lines, after transplantation of tooth germs to ectopic sites, and in organ culture. The in vivo approaches are the most physiological but do not provide accessibility to tooth germs for further experimental manipulation. Organ cultures, although readily accessible, do not sustain full tooth germ development and are appropriate for short‐term analysis. Thus, we sought to establish a new approach that would combine experimental accessibility with sustained development. We implanted fragments of embryonic day 12 mouse embryo first branchial arch containing early bud stage tooth germs into the lateral mesenchyme of day 4–5 chick embryo wing buds in ovo. Eggs were reincubated, and implanted tissues were examined by histochemistry and in situ hybridization over time. The tooth germs underwent seemingly normal growth, differentiation, and morphogenesis. They reached the cap, bell, and crown stages in approximately 3, 6, and 10 days, respectively, mimicking in a striking manner native temporal patterns. To examine mechanisms regulating tooth germ development, we first implanted tooth germ fragments, microinjected them with neutralizing antibodies to the key signaling molecule Sonic hedgehog (Shh), and examined them over time. Tooth germ development was markedly delayed, as revealed by poor morphogenesis and lack of mature ameloblasts and odontoblasts displaying characteristic traits such as an elongated cell shape, nuclear relocalization, and amelogenin gene expression. These phenotypic changes began to be reversed upon further incubation. The data show that the limb bud represents an effective, experimentally accessible as well as economical system for growth and analysis of developing tooth germs. The inhibitory effects of Shh neutralizing antibody treatment are discussed in relation to roles of this signaling pathway proposed by this and other groups previously. © 2002 Wiley‐Liss, Inc.
Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells
Authors: Conner Paul J. , Focke Paul J. , Noden Drew M. , Epstein Miles L.
Published: January 2003

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections. We studied the developing avian gut to characterize the pattern of migration of the crest cells, and the relationship between migration and differentiation. Crest cells at the leading edge of the migratory front appear as strands of cells; isolated individual crest cells are rarely seen. In the foregut and midgut, these strands are located immediately beneath the serosa. In contrast, crest cells entering the colon appear first in the deeper submucosal mesenchyme and later beneath the serosa. As the neural crest wavefront passes caudally, the crest cell cords become highly branched, forming a reticular lattice that presages the mature organization of the enteric nervous system. Neurons and glia first appear within the strands at the advancing wavefront. Later neurons are consistently located at the nodes where branches of the lattice intersect. In the most rostral foregut and in the colon, some neurons initially appear in close association with extrinsic nerve fibers from the vagus and Remak's nerve, respectively. We conclude that crest cells colonize the gut as chains of cells and that, within these chains, both neurons and glia appear close to the wavefront.© 2002 Wiley‐Liss, Inc.
Comparative genomic hybridization of 49 primary retinoblastoma tumors identifies chromosomal regions associated with histopathology, progression, and patient outcome
Authors: Lillington Debra M. , Kingston Judith E. , Coen Pietro G. , Price Elizabeth , Hungerford John , Domizio Paola , Young Bryan D. , Onadim Zerrin
Published: January 2003

Forty‐nine primary retinoblastoma (Rb) tumors were analyzed by the use of comparative genomic hybridization (CGH), and clinical/histological correlations were performed. Adverse histological factors were present in 13 patients. Chromosomal imbalance was a frequent phenomenon, seen in 96% of the tumors. Gain of 6p represented the most frequent event (69% of the tumors), whereas +1q was observed in 57%, confirming that these abnormalities are key secondary events in retinoblastoma tumor progression. Loss of 13q and 16 was significantly associated with tumors displaying adverse histo‐prognostic factors, whereas −16q was significantly associated with tumors without adverse features. In three patients who developed an extra‐ocular relapse, the tumors showed −13q and 2/3 had −5q, suggesting that these abnormalities may be associated with metastasis. Children ≥ 36 months of age at enucleation tended to have more CGH abnormalities per tumor than children < 12 months (median numbers 11 vs. 3). In addition, +1q, +13q, −16, and −16q were more frequent in children with an older age at enucleation. Identical CGH changes were found in both tumors from one patient with bilateral tumors, suggesting a common origin. It is possible that tumors displaying loss of 13q and 5q indicate those patients who may suffer an adverse outcome and who would require alternative or more intensive therapy. CGH analysis on larger cohorts and in prospective clinical trials will be invaluable in determining whether a genetic classification of retinoblastoma represents a reliable measure of prognosis. © 2003 Wiley‐Liss, Inc.
Hypermethylation‐mediated regulation of CD44 gene expression in human neuroblastoma
Authors: Yan Pu , Mühlethaler Annick , Bourloud Katia Balmas , Beck Maja Nenadov , Gross Nicole
Published: January 2003

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single‐strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44‐negative cell lines displayed hypermethylation in both regions, whereas all CD44‐expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44‐negative cells with the 5‐aza‐2′‐deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation‐independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell–specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation‐mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers. © 2003 Wiley‐Liss, Inc.
Chromosomes that show partial loss or gain in near‐diploid tumors coincide with chromosomes that show whole loss or gain in near‐triploid tumors: Evidence suggesting the involvement of the same genes in the tumorigenesis of high‐ and low‐risk neuroblastomas
Authors: Tomioka Nobumoto , Kobayashi Hirofumi , Kageyama Hajime , Ohira Miki , Nakamura Yohko , Sasaki Fumiaki , Todo Satoru , Nakagawara Akira , Kaneko Yasuhiko
Published: January 2003

We performed two‐color fluorescence in situ hybridization analysis to detect the numbers of chromosomes 1 and 17, 1p deletion, and 17q gain in 177 neuroblastomas, including 101 tumors that were found by a mass‐screening program for infants. Sixty‐eight tumors with disomy 1 or tetrasomy 1 were classified as the Dis1 group, and 109 tumors with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 were classified as the Tris1 group. 17q gain was the most frequent genetic event, followed by 1p deletion, and MYCN amplification in both Dis1 and Tris1 tumors. However, the incidence of all the genetic events was higher in Dis1 tumors than in Tris1 tumors. These findings suggest that Tris1 tumors are more resistant to acquiring the genetic events than are Dis1 tumors. In addition, there was an accumulation of genetic events in more advanced stages, with the exception of a high incidence of 17q gain in the stage IVS Tris1 tumors. Comparative genomic hybridization analysis, which was performed in 59 of the 177 tumors, showed that chromosomes partially lost or gained in Dis1 tumors coincided with chromosomes totally lost or gained in Tris1 tumors. Dis1 and Tris1 tumors were considered to have near‐diploid/tetraploid and near‐triploid/pentaploid chromosome numbers, respectively. These findings suggest that the same tumor‐suppressor genes or oncogenes may be involved in the development and progression of both high‐ and low‐risk neuroblastomas, and that the ploidy state of the tumor plays a fundamental role in the heterogeneous behavior. © 2003 Wiley‐Liss, Inc.
Genomic anatomy of the specific reciprocal translocation t(15;17) in acute promyelocytic leukemia
Authors: Reiter Andreas , Sauβele Susanne , Grimwade David , Wiemels Joseph L. , Segal Mark R. , Lafage‐Pochitaloff Marina , Walz Christoph , Weisser Andreas , Hochhaus Andreas , Willer Andreas , Reichert Anja , Büchner Thomas , Lengfelder Eva , Hehlmann Rüdiger , Cross Nicholas C.P.
Published: January 2003

The genomic breakpoints in the t(15;17)(q22;q21), associated with acute promyelocytic leukemia (APL), are known to occur within three different PML breakpoint cluster regions (bcr) on chromosome 15 and within RARA intron 2 on chromosome 17; however, the precise mechanism by which this translocation arises is unclear. To clarify this mechanism, we (i) assembled the sequence of RARA intron 2, (ii) amplified and sequenced the genomic PML‐RARA junction sequences from 37 APL patients, and (iii) amplified and sequenced the reverse RARA‐PML genomic fusion in 29 of these cases. Three significant breakpoint microclusters within RARA intron 2 were identified, suggesting that sequence‐associated or structural factors play a role in the formation of the t(15;17). There was no evidence that the location of a breakpoint in PML had any relationship to the location of the corresponding breakpoint in RARA. Although some sequence motifs previously implicated in illegitimate recombinations were found in the microcluster regions, these associations were not significant. Comparison of forward and reverse genomic junctions revealed microhomologies, deletions, and/or duplications of either gene in all but one case, in which a complex rearrangement with inversion of the PML‐derived sequence was found. These findings are consistent with the hypothesis that the t(15;17) occurs by nonhomologous recombination of DNA after processing of the double‐strand breaks by a dysfunctional DNA damage‐repair mechanism. © 2003 Wiley‐Liss, Inc.
Distinct chromosomal aberrations in sinonasal mucosal melanoma as detected by comparative genomic hybridization
Authors: van Dijk Marcory , Sprenger Sandra , Rombout Paul , Marres Henri , Kaanders Johannes , Jeuken Judith , Ruiter Dirk
Published: January 2003

Sinonasal mucosal melanomas are the most frequent mucosal melanomas and arise from melanocytes located in the nasal cavity and the paranasal sinuses. The melanoma types, cutaneous melanoma, uveal melanoma, and mucosal melanoma, differ in etiology, geographic distribution, and clinical behavior. Genetic alterations have been previously studied in cutaneous and uveal melanomas but, to the best of our knowledge, not in mucosal melanomas. Comparative genomic hybridization (CGH) was performed on 14 routinely processed sinonasal mucosal melanomas. Furthermore, ploidy analysis was performed on 11 tumors to provide complementary data on the DNA index. The CGH profiles of sinonasal mucosal melanomas show remarkably consistent alterations: chromosome arm 1q is gained in all tumors and gains of 6p and 8q are present in 93 and 57%, respectively. Comparison of CGH data with both the common variants of cutaneous melanoma and uveal melanoma revealed that sinonasal mucosal melanomas harbor a distinct pattern of chromosomal abnormalities. Ploidy analysis also showed that diploid tumors exhibit gains of 1q and alterations of chromosome 6 (3 of 3 cases tested), whereas clear‐copy gains and high‐copy gains were seen only in triploid and tetraploid tumors (6 of 8 cases tested). This indicates that alteration of chromosomes 1 and 6 may precede polyploidization and formation of clear‐copy gains and high‐copy gains. © 2003 Wiley‐Liss, Inc.
The IGFI receptor gene: A molecular target for disrupted transcription factors
Authors: Werner Haim , Roberts Charles T.
Published: January 2003

The biological actions of the insulin‐like growth factors, IGFI and IGFII, are mediated by their activation of the IGFI receptor (IGFIR), a transmembrane heterotetramer linked to the RAS‐RAF‐MAPK and PI3K‐PKB/AKT signal transduction cascades. The IGFIR displays potent mitogenic, antiapoptotic, and transforming activities, and is a prerequisite for oncogenic transformation. A number of transcription factors have been identified that control the expression of this gene and therefore determine, to a significant extent, the proliferative status of the cell. The purpose of this review is to summarize data showing that, under normal physiological conditions, expression of the IGFIR is under inhibitory control by a family of negative growth regulators or tumor suppressors. Cells with a reduced number of cell‐surface receptors are unable to progress through the cell cycle and remain in a postmitotic state. Loss‐of‐function mutation of tumor suppressors in certain cancers results in transcriptional derepression of the IGFIR gene, with ensuing increases in the levels of IGFIR and increased proliferative capacity. Understanding the molecular mechanisms responsible for transcriptional regulation of the IGFIR gene will prove important in designing novel therapies aimed at targeting the IGF axis. © 2003 Wiley‐Liss, Inc.
High‐resolution genomic profiling of occult micrometastatic tumor cells
Authors: Kraus Jürgen , Pantel Klaus , Pinkel Daniel , Albertson Donna G. , Speicher Michael R.
Published: January 2003

Metastasis is responsible for most deaths from cancer. Currently, little is known about the early genetic events in the metastatic evolution. Here we describe the application of a newly developed strategy for an in‐depth characterization of genomic changes in micrometastatic cells. Unique tumor cell lines were established from bone marrow of patients with cancer of the prostate and analyzed by multiplex‐FISH (M‐FISH) and array CGH. M‐FISH revealed that the occult disseminated cells were characterized by very complex numerical and structural aberrations. Many of these aberrations resulted in chromosomal gains and losses, such as losses of 8p, 13q, and 18q and gains of 8q, 9q, 20, and the X chromosome, which are typically observed in prostate cancer. Array CGH allowed an unprecedented high‐resolution assessment of copy number changes, pinpointing commonly gained or lost regions, which should narrow down the identification of regions critically involved in metastasis. Thus, occult micrometastatic cells are now amenable to detailed analyses of their genome. Markers for prognosis and treatment decisions can now be established. © 2003 Wiley‐Liss, Inc.