Tilapia sex determination: Where temperature and genetics meet

This review deals with the complex sex determining system of Nile tilapia, Oreochromis niloticus, governed by the interactions between a genetic determination and the influence of temperature, shown in both domestic and wild populations. Naturally sex reversed individuals are strongly suggested in two wild populations. This can be due to the masculinising temperatures which some fry encounter during their sex differentiation period when they colonise shallow waters, and/or to the influence of minor genetic factors. Differences regarding a) thermal responsiveness of sex ratios between and within Nile tilapia populations, b) maternal and paternal effects on temperature dependent sex ratios and c) nearly identical results in offspring of repeated matings, demonstrate that thermosensitivity is under genetic control. Selection experiments to increase the thermosensitivity revealed high responses in the high and low sensitive lines. The high-line showed ~ 90% males after 2 generations of selection whereas the weakly sensitive line had 54% males. This is the first evidence that a surplus of males in temperature treated groups can be selected as a quantitative trait. Expression profiles of several genes (Cyp19a, Foxl2, Amh, Sox9a,b) from the gonad and brain were analysed to define temperature action on the sex determining/differentiating cascade in tilapia. The coexistence of GSD and TSD is discussed.

The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle

The mammalian Cutl1 gene codes for the CCAAT displacement protein (CDP), which has been implicated as a transcriptional repressor in diverse processes such as terminal differentiation, cell cycle progression, and the control of nuclear matrix attachment regions. To investigate the in vivo function of Cutl1, we have replaced the C-terminal Cut repeat 3 and homeodomain exons with an in-frame lacZ gene by targeted mutagenesis in the mouse. The CDP-lacZ fusion protein is retained in the cytoplasm and fails to repress gene transcription, indicating that the Cutl1(lacZ) allele corresponds to a null mutation. Cutl1 mutant mice on inbred genetic backgrounds are born at Mendelian frequency, but die shortly after birth because of retarded differentiation of the lung epithelia, which indicates an essential role of CDP in lung maturation. A less pronounced delay in lung development allows Cutl1 mutant mice on an outbred background to survive beyond birth. These mice are growth-retarded and develop an abnormal pelage because of disrupted hair follicle morphogenesis. The inner root sheath (IRS) is reduced, and the transcription of Sonic hedgehog and IRS-specific genes is deregulated in Cutl1 mutant hair follicles, consistent with the specific expression of Cutl1 in the progenitors and cell lineages of the IRS. These data implicate CDP in cell-lineage specification during hair follicle morphogenesis, which resembles the role of the related Cut protein in specifying cell fates during Drosophila development.

Cancer and inflammation: differentiation by USPIO-enhanced MR imaging.

PURPOSE To assess ultrasmall superparamagnetic iron oxide particles (USPIO) -enhanced MR imaging for the differentiation of malignant from benign, inflammatory lesions. MATERIALS AND METHODS In this study, approved by the local animal care committee, VX2 carcinoma and intramuscular abscesses were implanted into the hind thighs of New Zealand White rabbits. MR imaging was performed pre contrast and serially for 24 h after the injection of USPIO. MR findings were compared with histopathologic results based on Prussian blue stains for the presence of iron. RESULTS Twenty-four hours after the Ferumoxtran-injection, no changes were observed in VX2 carcinomas, whereas a mean reduction of the contrast-to-noise ratio (CNR) of approximately 90% was noticed in abscesses as well as in necrotic tumors. On histopathologic examination, abscess and necrotic parts of the tumor were found to include iron-containing monocytes demonstrating that the reduction in CNR was caused by USPIO-tagged monocytes. CONCLUSION Our results prove the ability of USPIO-enhanced MRI to differentiate benign, inflammatory from malignant lesions.

Differentiation between active and chronic Crohn's disease using MRI small-bowel motility examinations — Initial experience

AIM: To evaluate the influence of locally active Crohn's disease on systemic small-bowel motility in patients with chronic Crohn's disease compared to healthy individuals. MATERIAL AND METHODS: Fifteen healthy individuals (11 men, four women; mean age 37 years) and 20 patients with histopathologically proven active (n = 15; 10 women, 5 men; mean age 45 years) or chronic (n = 5; four women, one man; mean age 48 years) Crohn's disease were included in this institutional review board-approved, retrospective study. Magnetic resonance imaging (MRI; 1.5 T) was performed after standardized preparation. Two-dimensional (2D) cine sequences for motility acquisition were performed in apnoea (27 s). Motility assessment was performed using dedicated software in three randomly chosen areas of the small-bowel outside known Crohn's disease-affected hotspots. The main quantitative characteristics (frequency, amplitude, occlusion rate) were compared using Student's t-test and one-way analysis of variance (ANOVA). RESULTS: Three randomly chosen segments were analysed in each participant. Patients with active Crohn's disease had significantly (p < 0.05) reduced contraction frequencies (active Crohn's disease: 2.86/min; chronic: 4.14/min; healthy: 4.53/min) and luminal occlusion rates (active: 0.43; chronic: 0.70; healthy: 0.73) compared to healthy individuals and patients with chronic Crohn's disease. Contraction amplitudes were significantly reduced during active Crohn's disease (6.71 mm) compared to healthy participants (10.14 mm), but this only reached borderline significance in comparison to chronic Crohn's disease (8.87 mm). Mean bowel lumen diameter was significantly (p = 0.04) higher in patients with active Crohn's disease (16.91 mm) compared to healthy participants (14.79 mm) but not in comparison to patients with chronic Crohn's disease (13.68). CONCLUSION: The findings of the present study suggest that local inflammatory activity of small-bowel segments in patients with active Crohn's disease alters small-bowel motility in distant, non-affected segments. The motility patterns revealed reduced contraction-wave frequencies, amplitudes, and decreased luminal occlusion rates. Thus evaluation of these characteristics potentially helps to differentiate between chronic and active Crohn's disease.

Characterization and differentiation of body fluids, putrefaction fluid, and blood using Hounsfield unit in postmortem CT

The purpose of the present study was to evaluate the ranges of Hounsfield unit (HU) found in body fluids, putrefaction fluids, and blood on postmortem CT and how these ranges are affected by postmortem interval, temperatures, and CT beam energy. Body fluids, putrefaction fluids, and blood from a total of 53 corpses were analyzed to determine the ranges of HU values from postmortem CT images that were taken prior to autopsy. The fluids measured in CT images were obtained at autopsy and examined in terms of macroscopic and microscopic appearances. Body fluids and blood were also collected in plastic bottles, which were subjected to CT scans at different beam energies (80-130 kV) and at various fluid temperatures (4 to 40 °C). At a postmortem interval of 1 to 4 days, the ranges of HU values of the serous fluids (13-38 HU) and the nonsedimented blood (40-88 HU) did not overlap. In the sedimented blood, the upper serum layer exhibited HU value ranges that overlapped with those of the serous fluids. The putrefaction fluids exhibited a range of HU values between 80 and -130 HU. Elevated HU values were observed in fluids with accretive cell impurities. HU values decreased slightly with increasing temperature and CT beam energy. We concluded that serous fluids and blood in fresh corpses can be characterized and differentiated from each other based on HU value ranges. In contrast, body fluids in decomposed corpses cannot be differentiated by their HU value ranges. Different beam energies and corpse temperatures had only minor influences on HU value ranges and therefore should not be obstacles to the differentiation and characterization of body fluids and blood.

[Computer tomographic differentiation of intrathoracic neoplasms and inflammation in the dog].

The purpose of this study was to elaborate on the advantages and limits of computed tomography (CT) in the differentiation of thoracic lesions, in particular neoplasias. In the course of the investigation CT-scans of the thorax of 31 dogs with lesions in the area of the lungs or mediastinum were evaluated. The lesions were rated by morphology, distribution pattern, attenuation values and contrast-enhancement. Biopsies or the whole body underwent a pathohistological examination. Of the 31 dogs 17 had neoplastic and 14 had inflammatory lesions in the thoracic region. With help of the CT, the exact localisations of the different lesions was possible in most cases. Due to their characteristic morphologies, distribution patterns and attenuation values the differentiation between inflammatory and neoplastic lesions was possible in most cases (n=25/31) on the basis of the CT-scans. Mean non-enhanced CT attenuation values of the neoplastic lesions ranged between 31 and 50 HU, of the inflammatory lesions between -251 and 9 HU. Both neoplastic and inflammatory lesions showed contrast enhancement (between 14 and 38 HU and between 2 and 95 HU respectively). The mediastinal abcesses enhanced mainly on the periphery of the lesion. A differentiation of the various types of neoplastic lesions based on the non-enhanced attenuation values was not successful. Only metastasis could be differentiated because of their distribution pattern.

Functional differentiation of spider hemocytes by light and transmission electron microscopy, and MALDI-MS-imaging

The most abundant cell types in the hemolymph of Cupiennius salei are plasmatocytes (70–80%) and granulocytes (20–30%). Both cells differ in shape, cytochemical and transmission electron microscopy staining of their cytoplasma and granules. According to MALDI-IMS (matrix-assisted laser desorption ionization mass spectrometry imaging), granulocytes exhibit ctenidin 1 (9510 Da) and ctenidin 3 (9568 Da), SIBD-1 (8675 Da), and unknown peptides with masses of 2207 and 6239 Da. Plasmatocytes exhibit mainly a mass of 6908 Da. Unknown peptides with masses of 1546 and 1960 Da were detected in plasmatocytes and granulocytes. Transmission electron microscopy confirms the presence of two compounds in one granule and cytochemical staining (light microscopy) tends to support this view. Two further hemocyte types (cyanocytes containing hemocyanin and prehemocytes as stem cells) are only rarely detected in the hemolymph. These four hemocyte types constitute the cellular part of the spider immune system and this is discussed in view of arachnid hemocyte evolution.

Postmortem MR quantification of the heart for characterization and differentiation of ischaemic myocardial lesions.

OBJECTIVES Recently, an MRI quantification sequence has been developed which can be used to acquire T1- and T2-relaxation times as well as proton density (PD) values. Those three quantitative values can be used to describe soft tissue in an objective manner. The purpose of this study was to investigate the applicability of quantitative cardiac MRI for characterization and differentiation of ischaemic myocardial lesions of different age. MATERIALS AND METHODS Fifty post-mortem short axis cardiac 3 T MR examinations have been quantified using a quantification sequence. Myocardial lesions were identified according to histology and appearance in MRI images. Ischaemic lesions were assessed for mean T1-, T2- and proton density values. Quantitative values were plotted in a 3D-coordinate system to investigate the clustering of ischaemic myocardial lesions. RESULTS A total of 16 myocardial lesions detected in MRI images were histologically characterized as acute lesions (n = 8) with perifocal oedema (n = 8), subacute lesions (n = 6) and chronic lesions (n = 2). In a 3D plot comprising the combined quantitative values of T1, T2 and PD, the clusters of all investigated lesions could be well differentiated from each other. CONCLUSION Post-mortem quantitative cardiac MRI is feasible for characterization and discrimination of different age stages of myocardial infarction. KEY POINTS • MR quantification is feasible for characterization of different stages of myocardial infarction. • The results provide the base for computer-aided MRI cardiac infarction diagnosis. • Diagnostic criteria may also be applied for living patients.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

S100A1 and S100B expression patterns identify differentiation status of human articular chondrocytes.

Many studies in the field of cell-based cartilage repair have focused on identifying markers associated with the differentiation status of human articular chondrocytes (HAC) that could predict their chondrogenic potency. A previous study from our group showed a correlation between the expression of S100 protein in HAC and their chondrogenic potential. The aims of the current study were to clarify which S100 proteins are associated with HAC differentiation status and to provide an S100-based assay for measuring HAC chondrogenic potential. The expression patterns of S100A1 and S100B were investigated in cartilage and in HAC cultured under conditions promoting dedifferentiation (monolayer culture) or redifferentiation (pellet culture or BMP4 treatment in monolayer culture), using characterized antibodies specifically recognizing S100A1 and S100B, by immunohistochemistry, immunocytochemistry, Western blot, and gene expression analysis. S100A1 and S100B were expressed homogeneously in all cartilage zones, and decreased during dedifferentiation. S100A1, but not S100B, was re-expressed in pellets and co-localized with collagen II. Gene expression analysis revealed concomitant modulation of S100A1, S100B, collagen type II, and aggrecan: down-regulation during monolayer culture and up-regulation upon BMP4 treatment. These results strongly support an association of S100A1, and to a lesser extent S100B, with the HAC differentiated phenotype. To facilitate their potential application, we established an S100A1/B-based flow cytometry assay for accurate assessment of HAC differentiation status. We propose S100A1 and S100B expression as a marker to develop potency assays for cartilage regeneration cell therapies, and as a redifferentiation readout in monolayer cultures aiming to investigate stimuli for chondrogenic induction.