Mitotic patterns in the migrating lateral line cells of zebrafish embryos

The sense organs of the posterior lateral line system (neuromasts) are formed by a migrating primordium. In zebrafish, the primordium comprises approximately 100 cells at the onset of migration, and has deposited approximately 300 cells by the end of the process. Here, we report localized phases of mitotic activity and of mitotic quiescence within the migrating primordium. Quiescence in the leading region seems associated to the formation of a new prospective neuromast, whereas quiescence in the trailing region follows a wave of mitoses that synchronize trailing cells in G0/G1 phase, anticipating neuromast differentiation. Manipulating the size of the primordium does not lead to changes in the rate of cell proliferation. We also show that two mitoses often take place nearly synchronously in adjacent cells, suggestive of a determinate lineage. We conclude that proliferation in the migrating primordium follows a stereotyped pattern that closely anticipates the normal development of the system. Developmental Dynamics 238:1042–1051, 2009. © 2009 Wiley‐Liss, Inc.     

Cadherin-17:一种消化道腺癌诊断的有效标记

Cadherin-17也称肝-肠钙黏蛋白，它是一种钙依赖性跨膜糖蛋白，在肠上皮中参与细胞之间的黏附。以往的报道显示Cadherin-17表达在起源于胃、胰腺和肠道的腺癌中，而在其它肿瘤中的表达未有报道。作者使用组织芯片的方法对不同部位的正常组织和518例腺癌组织进行了Cadherin-17的免疫组化染色。结果显示：在正常组织中，     

Cell proliferation in the developing lateral line system of zebrafish embryos.

The sensory organs of the embryonic lateral line system are deposited by migrating primordia that originate in the otic region. Here, we examine the pattern of cell proliferation in the posterior lateral line system. We conclude that three phases of cell proliferation are involved in the generation of this system, separated by two phases of mitotic quiescence. The first phase corresponds to generalized proliferation during gastrulation, followed by a first period of quiescence that may be related to the determination of the lateral line precursor cells. A second phase of proliferation takes place in the placode and migrating primordium. This region is organized in annuli that correspond to the expression of proneural/neurogenic genes. A second period of quiescence follows, corresponding to deposition and differentiation of the sensory organs. The third period of proliferation corresponds to continued renewal of hair cells by division of support cells within each sensory organ.     

人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立

目的：建立定量检测脑脊液（CSF）及血清中S100蛋白的方法，探讨S100蛋白的检测在辅助诊断克－雅病（CJD）中的应用。方法：利用脑cDNA文库，经PCR获得了S100基因并克隆至原核表达载体pGEX-2T上，在大肠埃希菌中表达了谷胱甘肽－S－转移酶（GST）－S100融合蛋白；融合蛋白经亲和纯化后，免疫家兔，制备抗体；抗体经纯化后，用生物素（BNHS）标记，建立了可定量检测S100蛋白的生物素－亲和素系统ELISA方法，并初步用于临床脑脊液的检测中。结果：所表达的GST－S100蛋白相对分子质量约为35000，以其为抗原制备的S100特异性抗血清具有良好的免疫反应性。建立了定量检测脑脊液中S100蛋白的双抗体夹心ELISA方法，对3例“可能性的CJD”患者（14－3－3蛋白阳性）和15例无痴呆症状患者脑脊液进行检测，结果显示，3例CJD患者脑脊液S100含量均超过2．900μg/L,而在无痴呆症状患者组中14例患者脑脊液S100含量都低于0．180μg/L。对正常人和CJD患者血清进行检测，显示S100蛋白含量个体间差异很大。结论：所建立的方法可用于脑脊液中S100蛋白的检测，进一步扩大标本量有助于明确脑脊液中S100蛋白的检测在辅助诊断CJD中的价值。     

S-100 protein: Is it useful as a tumour marker in diagnostic immunocytochemistry?

A heterogeneous group of 159 tumours was studied for the presence of S-100 protein by the immunoperoxidase technique in order to determine whether this marker may be of value in facilitating immunocytochemical diagnosis. Among cases of melanocytic and pigmented lesions, S-100 was widely distributed and demonstrated the strongest degrees of reactivity. S-100 protein was identified in virtually all nerve sheath tumours such as schwannomas, neurofibromas, myxoid sheath nerve tumour and also in some tumours of controversial histogenesis such as granular cell tumours. The great majority of carcinomas did not express S-100, with only two cases of breast carcinoma displaying focal S-100 staining. In a miscellaneous group of tumours S-100 was demonstrated in chordomas, myoepitheliomas and Wilms' tumour with Schwann cell differentiation. Despite its presence in a wide array of cell types, S-100 protein continues to be an extremely useful marker especially for soft tissue and peripheral nervous system tumours.     

Common efferents to lateral line and labyrinthine hair cells in aquatic vertebrates

The axonal arborization of lateral line efferent neurones of fishes and urodeles is demonstrated using retrogradely transported horseradish peroxidase. Axon collaterals can be traced into the lateral line nerve and the sensory epithelia of the labyrinth. In fishes efferent somata and axon collaterals are restricted to the ipsilateral side, but they are bilaterally distributed in urodeles. Because of the widespread axonal branching the efferents are considered to have a more general effect, rather than to influence single maculae selectively.     

Cadherin-4 plays a role in the development of zebrafish cranial ganglia and lateral line system.

We previously reported that cadherin-4 (also called R-cadherin) was expressed by the majority of the developing zebrafish cranial and lateral line ganglia. Cadherin-4 (Cdh4) function in the formation of these structures in zebrafish was studied using morpholino antisense technology. Differentiation of the cranial and lateral line ganglia and lateral line nerve and neuromasts of the cdh4 morphants was analyzed using multiple neural markers. We found that a subset of the morphant cranial and lateral line ganglia were disorganized, smaller, with reduced staining, and/or with altered shape compared to control embryos. Increased cell death in the morphant ganglia likely contributed to these defects. Moreover, cdh4 morphants had shorter lateral line nerves and a reduced number of neuromasts, which was likely caused by disrupted migration of the lateral line primordia. These results indicate that Cdh4 plays a role in the normal formation of the zebrafish lateral line system and a subset of the cranial ganglia.     

HOXB13 is a sensitive and specific marker of prostate cells, useful in distinguishing between carcinomas of prostatic and urothelial origin

The origin of a primary or metastatic carcinoma in the pelvic area is sometimes difficult to establish, in particular the distinction between those originating in the bladder and the prostate. A candidate marker is the HOXB13 gene, essential for prostate development. Some studies have shown expression of HOXB13 protein by immunohistochemistry in the nuclear compartment of benign prostate luminal epithelium and prostate carcinoma. Forty-two cases of biopsies and resection specimens of the prostate and urinary bladder, metastatic lymph nodes, and pelvic masses were retrieved from our databases. In all cases, doubt persisted regarding prostatic versus urothelial origin. All cases were stained for CK7, p63, p504s, PSA, CK20, and HOXB13. Chromogranin A, CD56, and synaptophysin were used when neuroendocrine differentiation was suspected. HOXB13 staining was negative or only weakly positive in all carcinomas of urothelial origin. Three of four carcinomas with neuroendocrine differentiation did not express HOXB13. The fourth carcinoma, in a patient with a history of prostate carcinoma, was positive. In two cases with a synchronous prostatic and urothelial carcinoma, HOXB13 was exclusively expressed in the prostatic carcinoma. Our results demonstrate that HOXB13 expression identifies prostatic origin of a carcinoma with good sensitivity (89 %) and very good specificity (100 %). HOXB13 is a specific and sensitive marker for prostate cells and a valuable diagnostic tool, especially when poorly differentiated or neuroendocrine tumors are encountered. These results justify testing of HOXB13 as a prostate-specific carcinoma marker in larger cohorts for a more thorough evaluation of its sensitivity and specificity.     

S100A9 a new marker for monocytic human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that negatively regulate the immune response during tumour progression, inflammation and infection. Only limited data are available on human MDSC because of the lack of specific markers. We have identified members of the S100 protein family-S100A8, S100A9 and S100A12 - specifically expressed in CD14(+) HLA-DR(-/low) MDSC. S100A9 staining in combination with anti-CD14 could be used to identify MDSC in whole blood from patients with colon cancer. An increase in the population of CD14(+) S100A9(high) MDSC was observed in the peripheral blood from colon cancer patients in comparison with healthy controls. Finally, nitric oxide synthase expression, a hallmark of MDSC, was induced in CD14(+) S100A9(high) upon lipopolysaccharide/interferon-γ stimulation. We propose S100 proteins as useful markers for the analysis and further characterization of human MDSC.     

Accuracy of the determination of S100 protein expression in malignant melanoma using a polyclonal antibody directed against S100 and monoclonal antibodies specific for S100 alpha and beta

Abstract

S100 proteins are present in a variety of tissues and perform regulatory functions in numerous metabolic processes. They have an important role in many human cancers, including malignant melanoma. Both polyclonal and monoclonal antibodies have been used to investigate S100 expression in melanoma tissue sections. This study aimed to determine the accuracy and sensitivity of these two types of antibodies in detecting S100 proteins in paraffin processed tissue cases of malignant melanoma. The study compared routinely used rabbit polyclonal anti-S100 antibody raised against both anti-S100A and B isoforms (Dako, Glostrup, Denmark), as per studies by Timar, and compared and contrasted findings with mouse monoclonal anti-S100A and anti-S100B antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The study involved the assessment of formalin-fixed paraffin-embedded tissue blocks from 56 cases of malignant melanoma, consisting of 23 superficial spreading, nine nodular, eight lentigo maligna, five acral lentigenous forms, five metastatic melanomas (two sentinel lymph node positive cases and three cases of nodal involvement from cases of elective nodal groin dissections), and six cases of desmoplastic malignant melanoma (DMM). The slides were stained by immunohistochemical methods on an automated platform (BenchMark XT; Roche, USA) and employing the iView detection system. All slides were examined by routine light microscopy by two independent assessors. The best results for both intensity of staining and percentage of positive tumor cells were achieved with polyclonal anti-S100 antibody and monoclonal anti-S100B antibody. Anti-S100A antibody yielded weaker staining intensity (with mean intensity of 1·8, compared to 2·8 for both anti-S100B antibody and polyclonal anti-S100 antibody), and a lower percentage of positive melanoma cells (an average of 74% for anti-S100A, compared to 95% for both anti-S100B antibody and polyclonal anti-S100 antibody). This result was statistically significant (P<0·01). Staining in cases of DMM gave the same results (P<0·01). The conclusion from this study is that polyclonal anti-S100 antibody and monoclonal anti-S100B antibody are more suitable than monoclonal anti-S100A antibody for diagnostic investigations of malignant melanoma, irrespective of the histological type of melanoma.     

Topographical and drug specific sensitivity of hair cells of the zebrafish larvae to aminoglycoside-induced toxicity

The hair cells of the lateral line system of fishes are morphologically and physiologically similar to the hair cells of the mammalian inner ear, also sharing its molecular characteristics. For this reason, it has been used as a powerful animal model to analyze in vivo ototoxicity. In this work, we examined the dose-dependent effects of two potent ototoxic aminoglycosides, neomycin and gentamicin, on the hair cells of two selected neuromasts (L1 and T1, the first of the trunk and the terminal located in the fin, respectively) of the lateral line in the ET4 transgenic zebrafish line. The hair cells of this strain selectively and constitutively display fluorescence. The fish were treated for 24 h at different doses (1, 2.5, 5, 10 and 100 μM levels) of both aminoglycosides. Immediately after treatment the morphology and the number of cells in L1 and T were analyzed under a fluorescence microscope. The results show that neomycin and gentamicin have different effects on the hair cell death at the same concentration, showing also different toxicity in L1 and T1 neuromasts. The toxicity observed in the hair cells of T1 neuromast was less than in L1 especially for the gentamicin treatment. These results demonstrate different sensitivity of hair cells of the lateral line to ototoxic drugs according to topographical localization and suggest the in vivo assay of the L1 neuromast of zebrafish larva and low doses of neomycin as an ideal model to study ototoxicity induced by aminoglycosides.     

Cadherin-2 function in the cranial ganglia and lateral line system of developing zebrafish.

Cadherins are cell surface molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin-2 (also called N-cadherin), a member of classic cadherin subfamily, has been shown to play important roles in development of a variety of tissues and organs, including the nervous system. We recently reported that cadherin-2 was strongly expressed by the majority of cranial ganglia and lateral line system of developing zebrafish. To gain insight into cadherin-2 role in the formation of these structures, we have used several markers to analyze zebrafish embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO). We find that development of several cranial ganglia, including the trigeminal, facial, and vagal ganglia, and the lateral line ganglia and neuromasts of the cdh2MO-injected embryos are severely disrupted. These phenotypes were confirmed by analyzing a cadherin-2 mutant, glass onion. Our results suggest that cadherin-2 function is crucial for the normal formation of the zebrafish lateral line system and a subset of cranial ganglia.     

Podoplanin is a useful marker for identifying mesothelioma in malignant effusions

The diagnosis of malignant mesothelioma in serosal effusions continues to be a major challenge because some of its cytomorphological features closely resemble adenocarcinomas. Immunohistochemistry is a valuable tool in the differentiation of epithelioid mesothelioma from metastatic adenocarcinomas. However, no single antibody has demonstrated absolute sensitivity or specificity. In this study, we evaluated the value of immunostaining pattern for podoplanin to differentiate mesothelioma from adenocarcinomas of various origins. Cell blocks from previously collected paraffin‐embedded cell blocks of 86 effusions (18 mesothelioma, 35 reactive mesothelium, 9 breast adenocarcinoma, 14 ovarian adenocarcinoma, and 10 lung adenocarcinoma) were retrieved from the file of the Department of Pathology at University of Michigan and Lund University in Sweden and were used for the study. Slides prepared from the cell blocks were stained for podoplanin. The percentage of immunostained cells was recorded as follows: 1+ (5–25%), 2+ (26–50%), and 3+ (>50%). A stain result involving <5% of cells was considered negative. The intensity of positive results was evaluated as strong, moderate, or weak. Podoplanin is expressed in 94% of malignant mesothelioma cases (17/18), 97% (30/31) of cases of reactive mesothelial, 0% of lung adenocarcinoma cases (0/9), 0% of breast adenocarcinoma (0/9), and 7% of ovarian adenocarcinoma (1/14). All positive cases of malignant mesothelioma and reactive mesothelium showed strong membranous reactivity to podoplanin. The one positive case of ovarian adenocarcinoma showed a weak membranous podoplanin immunostaining. On the basis of our results and published data, we believe that membranous podoplanin immunoreactivity, in conjunction with calretinin, would be more specific than CK5/6 and WT‐1 in differentiating epithelioid malignant mesothelioma from adenocarcinoma of the lung, breast, and ovary. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

S100A11 is a potential prognostic marker for clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is one of few cancers with rising incidence in North America. The prognosis of ccRCC is variable and difficult to predict. Stratification of patients according to disease aggressiveness can significantly improve patient management. We investigated the expression of the S100A11 protein in 385 patients with primary ccRCC using immunohistochemistry on tissue microarrays. We compared its expression with clinicopathologic parameters and patients’ survival. We also validated our results at the mRNA level on an independent set from The Cancer Genome Atlas. As a dichotomous variable (low vs. high expression), there was a significant association between S100A11 expression and tumor grade, with higher expression associated with higher tumor grades (p < 0.001). High expression was also significantly more frequently seen in higher versus lower stages (56 vs. 28 %). In the univariate analysis, high S100A11 expression was associated with significantly shorter disease-free survival (DFS) (HR = 2.28; p = 0.001). This was maintained in the multivariate analysis (HR = 1.69; p = 0.042). Expression was not associated with overall survival (OS) (p = 0.10). Comparable results were obtained when S100A11 expression was analyzed as a trichotomous variable (low, moderate, or high expression). The Kaplan–Meier survival analyses showed that higher S100A11 expression was associated with statistically significant decrease in DFS (p < 0.001), but not OS (p = 0.1).     

Single‐cell analysis of somatotopic map formation in the zebrafish lateral line system

The zebrafish lateral line is a simple sensory system comprising a small number of neurons in addition to their sensory organs, the neuromasts. We have adopted this system as a model for single‐cell level analyses of topographic map formation and examined when and how the lateral line topographic map is established. Single‐neuron labeling demonstrated that somatotopic organization of the ganglion emerges by 54 hr postfertilization, but also that this initial map is not as accurate as that observed at 6 days postfertilization. During this initial stage, individual neurons exhibit extensively diverse behavior and morphologies. We identified leader neurons, the axons of which are the first to reach the tail, and later‐appearing axons that contribute to the initial map. Our data suggest that lateral line neurons are heterogeneous from the beginning of lateral line development, and that some of them are intrinsically fate determined to contribute to the somatotopic map. Developmental Dynamics 239:2058–2065, 2010. © 2010 Wiley‐Liss, Inc.