Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.     

Interplay between human microglia and neural stem/progenitor cells in an allogeneic co‐culture model

Experimental neural cell therapies, including donor neural stem/progenitor cells (NPCs) have been reported to offer beneficial effects on the recovery after an injury and to counteract inflammatory and degenerative processes in the central nervous system (CNS). The interplay between donor neural cells and the host CNS still to a large degree remains unclear, in particular in human allogeneic conditions. Here, we focused our studies on the interaction of human NPCs and microglia utilizing a co‐culture model. In co‐cultures, both NPCs and microglia showed increased survival and proliferation compared with mono‐cultures. In the presence of microglia, a larger subpopulation of NPCs expressed the progenitor cell marker nestin, whereas a smaller group of NPCs expressed the neural markers polysialylated neural cell adhesion molecule, A2B5 and glial fibrillary acidic protein compared with NPC mono‐cultures. Microglia thus hindered differentiation of NPCs. The presence of human NPCs increased microglial phagocytosis of latex beads. Furthermore, we observed that the expression of CD200 molecules on NPCs and the CD200 receptor protein on microglia was enhanced in co‐cultures, whereas the release of transforming growth factor‐β was increased suggesting anti‐inflammatory features of the co‐cultures. To conclude, the interplay between human allogeneic NPCs and microglia, significantly affected their respective proliferation and phenotype. Neural cell therapy including human donor NPCs may in addition to offering cell replacement, modulate host microglial phenotypes and functions to benefit neuroprotection and repair.     

Inertial motion capture in conjunction with an artificial neural network can differentiate the gait patterns of hemiparetic stroke patients compared with able-bodied counterparts

Clinical gait analysis has proven to reduce uncertainties in selecting the appropriate quantity and type of treatment for patients with neuromuscular disorders. However, gait analysis as a clinical tool is under-utilised due to the limitations and cost of acquiring and managing data. To overcome these obstacles, inertial motion capture (IMC) recently emerged to counter the limitations attributed to other methods. This paper investigates the use of IMC for training and testing a back-propagation artificial neural network (ANN) for the purpose of distinguishing between hemiparetic stroke and able-bodied ambulation. Routine gait analysis was performed on 30 able-bodied control subjects and 28 hemiparetic stroke patients using an IMC system. An ANN was optimised to classify the two groups, achieving a repeatable network accuracy of 99.4%. It is concluded that an IMC system and appropriate computer methods may be useful for the planning and monitoring of gait rehabilitation therapy of stroke victims.     

聚赖氨酸修饰丝素蛋白膜对神经干细胞生长和分化的影响

利用聚赖氨酸修饰丝素蛋白膜,观察其对神经干细胞(NSCs)生长及分化的影响,为中枢神经系统损伤修复材料的选择提供实验基础和理论依据。文中首先制备聚赖氨酸修饰的丝素蛋白膜,并通过核磁共振图谱和紫外-可见光谱进行验证。NSCs分别接种在单纯丝蛋白膜(Silk)、聚赖氨酸修饰的丝蛋白膜(Silk-PIL)和多聚赖氨酸(PLL)上进行培养,分别在1、3、5、7 d时用CCK-8检测NSCs增殖活性。在第7天时,用免疫荧光染色检测NSCs分化情况,Western blotting和TUNEL检测细胞凋亡水平,Real-time PCR检测脑源性神经营养因子(BDNF)mRNA水平。结果表明,核磁共振图谱和紫外-可见光谱证明聚赖氨酸成功地接枝到了丝素蛋白膜上,CCK-8检测显示:从第3天开始一直到第7天,NSCs在Silk-PIL上的增殖活性要显著高于Silk组(P0.05),而与PLL组无显著性差异(P0.05)。免疫荧光观察显示,NSCs在Silk-PIL上分化成神经元的细胞显著多于Silk组(P0.05),而与PLL组无显著性差异,3个组之间分化为星型胶质细胞的数量并无显著性差异。Western blotting和TUNEL检测结果表明Silk-PIL组NSCs凋亡程度显著小于Silk组(P0.05),但与PLL组无显著性差异(P0.05)。RT-PCR结果显示,NSCs在Silk-PIL和PLL组的BDNFmRNA表达水平显著高于Silk组(P0.05)。结果表明,聚赖氨酸修饰的丝素蛋白膜能够促进NSCs的增殖活性并减少NSCs细胞凋亡,同时促进NSCs向神经元方向分化,有望成为新型组织工程支架材料搭载NSCs移植修复中枢神经系统损伤。     

TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells. In the absence of TRIM32, neuroblasts differentiate slower and show gene expression profiles that are characteristic of immature cells. Interestingly, TRIM32 deficiency induces more neural progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated olfactory bulb neurons of TRIM32 knockout mice. These results highlight the function of the cell fate-determinant TRIM32 for a balanced activity of the adult neurogenesis process.     

An evolutionary hybrid cellular automaton model of solid tumour growth

We propose a cellular automaton model of solid tumour growth, in which each cell is equipped with a micro-environment response network. This network is modelled using a feed-forward artificial neural network, that takes environmental variables as an input and from these determines the cellular behaviour as the output. The response of the network is determined by connection weights and thresholds in the network, which are subject to mutations when the cells divide. As both available space and nutrients are limited resources for the tumour, this gives rise to clonal evolution where only the fittest cells survive. Using this approach we have investigated the impact of the tissue oxygen concentration on the growth and evolutionary dynamics of the tumour. The results show that the oxygen concentration affects the selection pressure, cell population diversity and morphology of the tumour. A low oxygen concentration in the tissue gives rise to a tumour with a fingered morphology that contains aggressive phenotypes with a small apoptotic potential, while a high oxygen concentration in the tissue gives rise to a tumour with a round morphology containing less evolved phenotypes. The tissue oxygen concentration thus affects the tumour at both the morphological level and on the phenotype level.     

Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling

Cellular biomolecular complexes including protein–protein, protein–RNA, and protein–DNA interactions regulate and execute most biological functions. In particular in brain, protein–protein interactions (PPIs) mediate or regulate virtually all nerve cell functions, such as neurotransmission, cell–cell communication, neurogenesis, synaptogenesis, and synaptic plasticity. Perturbations of PPIs in specific subsets of neurons and glia are thought to underly a majority of neurobiological disorders. Therefore, understanding biological functions at a cellular level requires a reasonably complete catalog of all physical interactions between proteins. An enzyme-catalyzed method to biotinylate proximal interacting proteins within 10 to 300 nm of each other is being increasingly used to characterize the spatiotemporal features of complex PPIs in brain. Thus, proximity labeling has emerged recently as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and PPIs in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. In this review, we summarize recent advances in proximity labeling methods and their application to neurobiology.     

Stem cell competition driven by the Axin2-p53 axis controls brain size during murine development

1. Download : Download high-res image (281KB)
2. Download : Download full-size image

     

Elimination of heparan sulfate by heparitinases induces abnormal mesodermal and neural formation in Xenopus embryos

The expression of heparan sulfate glycosaminoglycan (HS-GAG) was examined in Xenopus embryos during the developmental stages. Chemical analysis showed the existence of HS-GAG in the ³⁵S-labeled embryos. By western blot analysis using a specific anti-HS monoclonal antibody, HS-GAG related epitope was found after the neurulation on two protein bands, whose molecular weights were approximately 90 kDa and 100 kDa, respectively. Immunohistochemistry revealed that HS-GAG occurred exclusively in the animal hemisphere in early gastrulae, and then appeared predominantly on the sheath of the neural tube, the notochord and epithelium. To address whether HS-GAG chains contribute to Xenopus embryonic development, we eliminated the embryonic HS-GAG by injecting purified Flavobacterium heparitinases (HSase) into their blastocoels. Most of the injected embryos were aberrant in mesodermal and neural formation, and became acephalic. Histological examination showed that these embryos were completely devoid of the central nervous system and the mesodermal tissues. Neither heat-inactivated heparitinase nor chondroitinase showed such abnormality. The HS-GAG-eliminated embryos showed decreased expression of both muscular and neural-specific markers. These results suggest that HS-GAG plays an indispensable role in establishing the fundamental body plan during early Xenopus development.