Top2b is involved in the formation of outer segment and synapse during late-stage photoreceptor differentiation by controlling key genes of photoreceptor transcriptional regulatory network.

Topoisomerase II beta (Top2b) is an enzyme that alters the topologic states of DNA during transcription. Top2b deletion in early retinal progenitor cells causes severe defects in neural differentiation and affects cell survival in all retinal cell types. However, it is unclear whether the observed severe phenotypes are the result of cell-autonomous/primary defects or non-cell-autonomous/secondary defects caused by alterations of other retinal cells. Using photoreceptor cells as a model, we first characterized the phenotypes in Top2b conditional knockout. Top2b deletion leads to malformation of photoreceptor outer segments (OSs) and synapses accompanied by dramatic cell loss at late-stage photoreceptor differentiation. Then, we performed mosaic analysis with shRNA-mediated Top2b knockdown in neonatal retina using in vivo electroportation to target rod photoreceptors in neonatal retina. Top2b knockdown causes defective OS without causing a dramatic cell loss, suggesting a Top2b cell-autonomous function. Furthermore, RNA-seq analysis reveals that Top2b controls the expression of key genes in the photoreceptor gene-regulatory network (e.g., Crx, Nr2e3, Opn1sw, Vsx2) and retinopathy-related genes (e.g., Abca4, Bbs7, Pde6b). Together, our data establish a combinatorial cell-autonomous and non-cell-autonomous role for Top2b in the late stage of photoreceptor differentiation and maturation. © 2017 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Study on heavy metal levels and its health risk assessment in some edible fishes from Nansi Lake, China.

Eight heavy metals, namely Cu, Zn, Fe, Mn, Cd, Ni, Pb, and As in the muscles of nine fish species collected from Nansi Lake, China. were determined, and the potential health risks to local residents via consumption of the fishes were estimated. The results of two-way ANOVA that showed the concentrations of heavy metals in the investigated fish samples were influenced significantly by fish species and sampling sites. Correlation analysis indicated that sampling sites had significant effects on the levels of correlation coefficients among different heavy metal concentrations. Interestingly, although none of the hazard quotient (HQ) values of any individual element was greater than 1 for the investigated exposure population through fish consumption, the hazard index (HI) values were more than 1 for local fishermen, suggesting that local fishermen may be experiencing some adverse health effects. Among the investigated nine fish species, Cyprinus carpio had the highest HQ and HI. As, Pb, and Cd were the most concerning heavy metals in the investigated fish samples due to their higher relative contributions to the HI values.

Identification of a transient Sox5 expressing progenitor population in the neonatal ventral forebrain by a novel cis-regulatory element.

Precise control of lineage-specific gene expression in the neural stem/progenitor cells is crucial for generation of the diversity of neuronal and glial cell types in the central nervous system (CNS). The mechanism underlying such gene regulation, however, is not fully elucidated. Here, we report that a 377 bp evolutionarily conserved DNA fragment (CR5), located approximately 32 kbp upstream of Olig2 transcription start site, acts as a cis-regulator for gene expression in the development of the neonatal forebrain. CR5 is active in a time-specific and brain region-restricted manner. CR5 activity is not detected in the embryonic stage, but it is exclusively in a subset of Sox5+ cells in the neonatal ventral forebrain. Furthermore, we show that Sox5 binding motif in CR5 is important for this cell-specific gene regulatory activity; mutation of Sox5 binding motif in CR5 alters reporter gene expression with different cellular composition. Together, our study provides new insights into the regulation of cell-specific gene expression during CNS development.

Heavy metals in jujubes and their potential health risks to the adult consumers in Xinjiang province, China.

The concentrations of seven heavy metals (HMs) in jujube samples collected from Hetian region (HTR), Hami region (HMR), Erkesu region (ESR), Bayikuleng region (BLR), and Turpan region (TUR) were determined by inductively coupled mass spectrometry (ICP-MS). The accuracy and precision of the analytical method were confirmed by the certified reference material (GBW 07605). In general, the concentration of iron was higher than those of the other six metals in the investigated jujube samples. The Hazard Quotient (HQ) and Hazard Index (HI) were calculated to evaluate the noncarcinogenic health risk from individual metal and combined metals due to the dietary intakes via consumption of jujube. Both HQ and HI levels were far below 1, suggesting no noncarcinogenic risks for Xinjiang adults under the current consumption rates of the jujubes. Among the jujubes from five different regions, BLR jujube had the highest HQ and HI. Fe and As were the most concerning HMs in the investigated jujube samples due to their higher relative contributions to HIs.

Topoisomerase IIbeta is required for proper retinal development and survival of postmitotic cells.

Topoisomerase IIbeta (Top2b) is an enzyme that modulates DNA supercoiling by catalyzing the passage of DNA duplexes through one another. It is ubiquitously expressed in postmitotic cells and known to function during the development of neuromuscular junctions in the diaphragm and the proper formation of laminar structure in the cerebral cortex. However, due to the perinatal death phenotype of the traditional constitutive and brain-specific Top2b knockout mice, the precise in vivo function of Top2b, especially during postnatal neural development, remains to be determined. Using both the constitutive and retina-specific knockout mouse models, we showed that Top2b deficiency resulted in delayed neuronal differentiation, degeneration of the plexiform layers and outer segment of photoreceptors, as well as dramatic reduction in cell number in the retina. Genome-wide transcriptome analysis by RNA sequencing revealed that genes involved in neuronal survival and neural system development were preferentially affected in Top2b-deficient retinas. Collectively, our findings have indicated an important function of Top2b in proper development and the maintenance/survival of postmitotic neurons in the retina.

A cis-element in the Notch1 locus is involved in the regulation of gene expression in interneuron progenitors.

Interneurons comprise approximately one third of the total cortical neurons in the mammalian cerebral cortex. Studies have revealed many details in the generation of this cell type. However, the mechanism that defines interneuron-lineage specific gene expression is not well understood. Gene regulatory elements, e.g., promoters, enhancers, and trans-acting factors, are essential for the proper control of gene expression. Here, we report that a novel evolutionarily conserved cis-element in the second intron of the Notch1 locus plays an important role in regulating gene expression in interneuron progenitors. The spatiotemporal activity of the cis-element in the developing central nervous system (CNS) was determined by both transient reporter expression in the developing chick and a transgenic mouse model. Its activity is well correlated with neurogenesis in both the chick and mouse and restricted to neural progenitor cells in the ganglionic eminence that are fated to differentiate into GABAergic interneurons of the neocortex. We further demonstrate that the cis-element activity requires the binding motif for trans-acting factors Gsh1/Barx2/Brn3. Deletion of this binding motif abolishes reporter gene expression. Together, these data provide new insights into the regulatory mechanisms of interneuron development in the vertebrate CNS.

Assessment of heavy metals in some wild edible mushrooms collected from Yunnan Province, China.

Eight heavy metals (Cu, Zn, Fe, Mn, Cd, Cr, Ni, and Pb) in 14 different wild-growing edible mushroom species (Coprinus comatus, Voluariella volvacea, Pleurotus nebrodensis, Hypsizigus marmoreus, Hericium erinaceus, Agrocybe aegerita, Lenfinus edodes, Collybia velutipes, Agaricus bisporus, Russula albida, Clitocybe conglobata, Pleurotus eryngii, Lepista sordida, and Pleurotus ostreatus) collected from Yunnan province, China, were determined by inductively coupled plasma-atomic emission spectrometry after microwave digestion. All element concentrations were determined on a dry weight basis. The ranges of element concentrations for copper, zinc, iron, manganese, cadmium, chromium, nickel, and lead were 6.8-31.9, 42.9-94.3, 67.5-843, 13.5-113, 0.06-0.58, 10.7-42.7, 0.76-5.1, and 0.67-12.9 mg/kg, respectively. In general, iron content was higher than other metals in all mushroom species. The levels of zinc, cadmium, and lead in some edible mushroom samples were found to be higher than legal limits. The relative standard deviations were found below 10%. The accuracy of procedure was confirmed by certified reference material.

Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.

BACKGROUND: Retinal cell development has been extensively investigated; however, the current knowledge of dynamic morphological and molecular changes is not yet complete. RESULTS: This study was aimed at revealing the dynamic morphological and molecular changes in retinal cell development during the embryonic stages using a new method of targeted retinal injection, in ovo electroporation, and immunohistochemistry techniques. A plasmid DNA that expresses the green fluorescent protein (GFP) as a marker was delivered into the sub-retinal space to transfect the chick retinal stem/progenitor cells at embryonic day 3 (E3) or E4 with the aid of pulses of electric current. The transfected retinal tissues were analyzed at various stages during chick development from near the start of neurogenesis at E4 to near the end of neurogenesis at E18. The expression of GFP allowed for clear visualization of cell morphologies and retinal laminar locations for the indication of retinal cell identity. Immunohistochemistry using cell type-specific markers (e.g., Visinin, Xap-1, Lim1+2, Pkcalpha, NeuN, Pax6, Brn3a, Vimentin, etc.) allowed further confirmation of retinal cell types. The composition of retinal cell types was then determined over time by counting the number of GFP-expressing cells observed with morphological characteristics specific to the various retinal cell types. CONCLUSION: The new method of retinal injection and electroporation at E3 - E4 allows the visualization of all retinal cell types, including the late-born neurons, e.g., bipolar cells at a level of single cells, which has been difficult with a conventional method with injection and electroporation at E1.5. Based on data collected from analyses of cell morphology, laminar locations in the retina, immunohistochemistry, and cell counts of GFP-expressing cells, the time-line and dynamic morphological and molecular changes of retinal cell development were determined. These data provide more complete information on retinal cell development, and they can serve as a reference for the investigations in normal retinal development and diseases.

Probing the influence of myelin and glia on the tensile properties of the spinal cord.

Although glia have been historically classified as the structurally supporting cells of the central nervous system, their role in tissue mechanics is still largely unstudied. The influence of myelin and glia on the mechanical properties of spinal cord tissue was examined by testing embryonic day 18 chick embryo spinal cords in uniaxial tension following disruption of the glial matrix using either ethidium bromide (EB) or an antibody against galactocerebroside (alphaGalC) in the presence of complement. Demyelination was confirmed by myelin basic protein immunoreactivity and quantified using osmium tetroxide staining. A substantial loss of astrocytes and oligodendrocytes concurrent with demyelination was observed following EB injection but not alphaGalC injection. No morphological changes were observed following injection of saline or IgG with complement as controls for EB and alphaGalC. Demyelinated spinal cords demonstrated significantly lower stiffness and ultimate tensile stress than myelinated spinal cords. No significant differences were observed in the tensile response between the two demyelinating protocols. The results demonstrate that the glial matrix provides significant mechanical support to the spinal cord, and suggests that myelin and cellular coupling of axons via the glial matrix in large part dictates the tensile response of the tissue.

Axon kinematics change during growth and development.

The microkinematic response of axons to mechanical stretch was examined in the developing chick embryo spinal cord during a period of rapid growth and myelination. Spinal cords were isolated at different days of embryonic (E) development post-fertilization (E12, E14, E16, and E18) and stretched 0%, 5%, 10%, 15%, and 20%, respectively. During this period, the spinal cord grew approximately 55% in length, and white matter tracts were myelinated significantly. The spinal cords were fixed with paraformaldehyde at the stretched length, sectioned, stained immunohistochemically for neurofilament proteins, and imaged with epifluorescence microscopy. Axons in unstretched spinal cords were undulated, or tortuous, to varying degrees, and appeared to straighten with stretch. The degree of tortuosity (ratio of the segment's pathlength to its end-to-end length) was quantified in each spinal cord by tracing several hundred randomly selected axons. The change in tortuosity distributions with stretch indicated that axons switched from non-affine, uncoupled behavior at low stretch levels to affine, coupled behavior at high stretch levels, which was consistent with previous reports of axon behavior in the adult guinea pig optic nerve (Bain, Shreiber, and Meaney, J. Biomech. Eng., 125(6), pp. 798-804). A mathematical model previously proposed by Bain et al. was applied to quantify the transition in kinematic behavior. The results indicated that significant percentages of axons demonstrated purely non-affine behavior at each stage, but that this percentage decreased from 64% at E12 to 30% at E18. The decrease correlated negatively to increases in both length and myelination with development, but the change in axon kinematics could not be explained by stretch applied during physical growth of the spinal cord. The relationship between tissue-level and axonal-level deformation changes with development, which can have important implications in the response to physiological forces experienced during growth and trauma.