Study of Hypoxia on Embryogenesis, Pharmaceutical Testing and Stem Cell Regulation Using Drosophila Model

Drosophila melanogaster has been used to study human disease as a model organism for many years. Many basic biological, physiological, and neurological properties are conserved between mammals and fly. We investigated its applications in the study of the impact of environmental stress on embryogenesis and its compensating mechanism, its uniqueness and powerfulness in modern pharmaceutical testing and screening, and its application in gene function identification. First we directly investigated Drosophila embryo development in vivo inside a customized microfluidic device with an established local oxygen gradient on a micrometer scale. When the embryos were placed in various conditions, two of the key developmental activities, the germ band shortening and the tail retraction, were examined during the embryogenesis. The time-lapse live cell imaging technique was used to monitor the cell morphology changes and pattern migration with the help of green fluorescence protein markers. Our results show that the examined activities during the Drosophila embryogenesis are highly sensitive to oxygen concentrations. Using this information, we presented a model to estimate the oxygen permeability across the Drosophila embryonic layers for the first time. Secondly, the Drosophila testis was used to evaluate the basic therapeutic mechanisms of the active components of several traditional Chinese medicines (TCM) that is known related to animal genital system and sexual function. Specifically, we investigated the effect of the compounds that were extracted from above-mentioned Chinese medicines on Drosophila germline stem cells (GSCs) by quantifying the GSCs mitotic activity and GSC number. Our results showed that, flies have a significantly higher cell cycle index when fed at certain concentration of icariin and Tanshinone IIA, the primary active component of YYH and DS, respectively. Other tested concentrations of extract produced either toxicity or insignificant effects on the mitotic activity. This indicates their function of promoting the GSCs mitosis. At last, we analyzed the expression and localization of two polarity genes throughout the cell cycle, and investigated how they affected mitotic spindle dynamics in asymmetric stem cell divisions. In stem cell divisions, it is critical to maintain tissue homeostasis by balancing the number of stem cells and progenitor cells. Improperly balancing may result in tumorigenesis due to tissue hyper-proliferation or tissue ageing due to tissue degeneration. Previous studies show that cyst stem cells (CySCs) in Drosophila testis divide asymmetrically. This behavior is ensured by the stem cell mitotic spindle repositioning, during which one of the spindle poles always moves close to the stem cell niche (a.k.a. hub cells) near the onsite of anaphase. Known as polarity proteins, the apically localized Par polarity complex, containing Bazooka (Baz; homolog of Par-3 in D. melanogaster), its binding target atypical Protein Kinase C (aPKC), and Par-6, are widely reported to be crucial in polarized cell epithelium and asymmetric cell division in multiple stem cell systems. We found that Baz and aPKC are required in Drosophila CySC asymmetric cell division

Pole cells move slower in hypoxia.

<p>Selected frames from a time-lapse analysis show α-tubulin green fluorescent protein (α-tub-GFP) <i>Drosophila</i> embryos are exposed to hypoxia <b>(A-F)</b> and normoxia <b>(A’-F’)</b>, respectively. The pole cells migration time is 37 min for a normoxic embryo and 137min for a hypoxic embryo. Stars indicate the center of pole cell clusters. Scale Bar: 100<i>μ</i>m. <b>(G)</b> Pole cells migration time under hypoxia and normoxia.</p

Parameters and values used in the simulation.

<p>Parameters and values used in the simulation.</p

Effect of localized hypoxia on <i>Drosophila</i> embryo development

<div><p>Environmental stress, such as oxygen deprivation, affects various cellular activities and developmental processes. In this study, we directly investigated <i>Drosophila</i> embryo development <i>in vivo</i> while cultured on a microfluidic device, which imposed an oxygen gradient on the developing embryos. The designed microfluidic device enabled both temporal and spatial control of the local oxygen gradient applied to the live embryos. Time-lapse live cell imaging was used to monitor the morphology and cellular migration patterns as embryos were placed in various geometries relative to the oxygen gradient. Results show that pole cell movement and tail retraction during <i>Drosophila</i> embryogenesis are highly sensitive to oxygen concentrations. Through modeling, we also estimated the oxygen permeability across the <i>Drosophila</i> embryonic layers for the first time using parameters measured on our oxygen control device.</p></div

<i>engrailed</i> stripe migration time under different oxygen levels.

<p><b>(A)</b> Data (red circles) showed every <i>engrailed</i> stripe migration time that measured at various oxygen conditions. The best-fit (least squares) curve (blue line) showed the delay on <i>engrailed</i> migration time when oxygen concentration dropped. <b>(B)</b> The extended best-fit curve (blue), combined with a horizontal asymptote line (dashed), showed the trend of the delay and the critical condition at 4.9% of oxygen, which could be the cutoff oxygen level that paused the germband shortening process.</p

<i>engrailed</i> stripe migration was arrested under hypoxia and quickly resumed upon switching to normoxia.

<p><b>(A)</b> An embryo with a fully developed <i>engrailed</i> pattern was positioned under hypoxia. <b>(B-D)</b> After 245 min of imaging, the stripe migration did not occur. <b>(E)</b> The oxygen condition was changed from hypoxia to normoxia. <b>(F)</b> The stripe migration was captured 12 min after the oxygen condition inside the device was changed. <b>(G, H)</b> The embryo had continuous stripe migration with normal speed. Images were taken every 3 min. Scale bar, 100 μm. Arrows indicate the 9^th stripe location under hypoxia (red) and normoxia (blue).</p

Experimental setup and the quantification methods.

<p><b>(A and A’)</b> An illustration depicting the microfluidic channels and the positioning of the embryos in a microfluidic device, which is designed to establish the oxygen concentration gradients through microchannels with different infused gases. <b>(B)</b> The oxygen concentration profile inside the microfluidic device was measured. The oxygen concentration gradients at 0 μm and 180 μm above the gas emitting surface are plotted using 20 points rolling average, shown as blue and red lines, respectively. <b>(C)</b> The pole cell migration time was measured as the pole cells (white plate) move from 10% to 90% of their total migration distance. <b>(D)</b> An illustration shows two ventral stripes (10^th and 11^th) pass through the posterior (reference point: *) of the embryo body with a counter-clockwise migration as indicated by the pink arrow. The time difference is defined as the <i>engrailed</i> stripe migration time. A: anterior; D: dorsal side; P: posterior; V: ventral side. Scale bar: 100 μm. <b>(E and F)</b> <i>engrailed</i> patterns (14 stripes) are shown before (E) and after (F) the tail retraction. The 9^th-11^th stripes are labeled indicating the stripe migration.</p

High-Performance All-Solid-State Polymer Electrolyte with Controllable Conductivity Pathway Formed by Self-Assembly of Reactive Discogen and Immobilized via a Facile Photopolymerization for a Lithium-Ion Battery

All-solid-state polymer electrolytes (SPEs) have aroused great interests as one of the most promising alternatives for liquid electrolyte in the next-generation high-safety, and flexible lithium-ion batteries. However, some disadvantages of SPEs such as inefficient ion transmission capacity and poor interface stability result in unsatisfactory cyclic performance of the assembled batteries. Especially, the solid cell is hard to be run at room temperature. Herein, a novel and flexible discotic liquid-crystal (DLC)-based cross-linked solid polymer electrolyte (DLCCSPE) with controlled ion-conducting channels is fabricated via a one-pot photopolymerization of oriented reactive discogen, poly­(ethylene glycol)­diacrylate, and lithium salt. The experimental results indicate that the macroscopic alignment of self-assembled columns in the DLCCSPEs is successfully obtained under annealing and effectively immobilized via the UV photopolymerization. Because of the existence of unique oriented structure in the electrolytes, the prepared DLCCSPE films exhibit higher ionic conductivities and better comprehensive electrochemical properties than the DLCCSPEs without controlled ion-conductive pathways. Especially, the assembled LiFePO₄/Li cells with oriented electrolyte show an initial discharge capacity of 164 mA h g^–1 at 0.1 C and average specific discharge capacities of 143, 135, and 149 mA h g^–1 at the C-rates of 0.5, 1, and 0.2 C, respectively. In addition, the solid cell also shows the first discharge capacity of 124 mA h g^–1 (0.2 C) at room temperature. The outstanding cell performance of the oriented DLCCSPE should be originated from the macroscopically oriented and self-assembled DLC, which can form ion-conducting channels. Thus, combining the excellent performance of DLCCSPE and the simple one-pot fabricating process of the DLC-based all-solid-state electrolyte, it is believed that the DLC-based electrolyte can be one of the most promising electrolyte materials for the next-generation high-safety solid lithium-ion batteries