In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart¹, retina², spinal cord³, optic nerve⁴, sensory hair cells⁵, and fins⁶.The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B)^6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)⁸. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)⁸. Depending on the level of the amputation, full regeneration is completed in a week to a month.The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration^9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists¹³, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated^7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development^17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.     

Response of wheat canopy CO2 and water gas-exchange to soil water content under ambient and elevated CO2

The nature of the interaction between drought and elevated CO₂ partial pressure (pC_a) is critically important for the effects of global change on crops. Some crop models assume that the relative responses of transpiration and photosynthesis to soil water deficit are unaltered by elevated pC_a, while others predict decreased sensitivity to drought at elevated pC_a. These assumptions were tested by measuring canopy photosynthesis and transpiration in spring wheat (cv. Minaret) stands grown in boxes with 100 L rooting volume. Plants were grown under controlled environments with constant light (300 µmol m^?2 s^?1) at ambient (36 Pa) or elevated (68 Pa) pC_a and were well watered throughout growth or had a controlled decline in soil water starting at ear emergence. Drought decreased final aboveground biomass (?15%) and grain yield (?19%) while elevated pC_a increased biomass (+24%) and grain yield (+29%) and there was no significant interaction. Elevated pC_a increased canopy photosynthesis by 15% on average for both water regimes and increased dark respiration per unit ground area in well‐watered plants, but not drought‐grown ones. Canopy transpiration and photosynthesis were decreased in drought‐grown plants relative to well‐watered plants after about 20–25 days from the start of the drought. Elevated pC_a decreased transpiration only slightly during drought, but canopy photosynthesis continued to be stimulated so that net growth per unit water transpired increased by 21%. The effect of drought on canopy photosynthesis was not the consequence of a loss of photosynthetic capacity initially, as photosynthesis continued to be stimulated proportionately by a fixed increase in irradiance. Drought began to decrease canopy transpiration below a relative plant‐available soil water content of 0.6 and canopy photosynthesis and growth below 0.4. The shape of these responses were unaffected by pC_a, supporting the simple assumption used in some models that they are independent of pC_a.     

细叶益母草(Leonurus sibiricus)叶表面腺毛多样性及发育形态学研究

本文研究了分布在细叶益母草（Ｌｅｏｎｕｒｕｓｓｉｂｉｒｉｃｕｓ） 叶表面三种腺毛的发育过程,在此基础上,对２细胞头状腺毛、４细胞头状腺毛和８细胞盾状腺毛的多样性特征进行了讨论     

Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

温度对红点唇瓢虫实验种群的影响

在７种温度下测定了红点唇瓢虫（Ｃｈｉｌｏｃｏｒｕｓ ｋｕｗａｎａｅ Ｓｉｌｖｅｓｔｒｉ）发育速率,并求得各虫态的发育起点温度和有效积温。其发育速率与温度的关系能很好地用王如松等（１９８２）提出的模型进行拟合。由此模型估计出最低、最高临界温度和最适发育温度,分别为１０．４２－１３．０１－℃、℃３３．５３－３７．０３℃和２４．９９－３０．１２℃。卵期忍耐温度变化的能力最强。４龄幼虫最弱。温度明显地影响     

Thermotactile thresholds at the fingertip: Effect of contact area and contact location

Thresholds for the detection of changes in temperature are used to indicate neuropathy, but a variety of different contact areas and contact locations are used. This study was designed to determine the effects of variations in contact area and contact location on both warm and cool thresholds at the fingertip. With 20 healthy subjects (10 females and 10 males aged 20–30 years), warm thresholds and cool thresholds were determined in two separate sessions using the method of limits. In the first part of each session, thresholds were determined around the centre of the whorl using circular contactors with five different diameters (3, 6, 9, 12, and 55 mm). In the second part of each session, thresholds were determined using two contactors (6- and 12-mm diameter) at three locations along the fingertip: (i) distal (5 mm from the nail), (ii) middle (centre of whorl), and (iii) proximal (3 mm from the distal interphalangeal joint). With increasing contact area, the warm thresholds decreased, the cool thresholds increased, and the inter-subject variability in both warm and cool thresholds decreased. Using the 6-mm diameter contactor, warm thresholds were independent of location but cool thresholds increased from distal to proximal locations. It is concluded that temperature sensitivity at the fingertip increases with increasing area of contact, with the variability in thresholds consistent with the existence of warm and cool “insensitive fields”. The findings show that the influence of contact area and contact location should be considered when assessing thermotactile thresholds at the fingertip.     

Interaction of the Neurospora crassa heat shock factor with the heat shock element during heat shock and different developmental stages

The interaction of the heat shock factor (HSF) with the heat shock element (HSE) was determined by a non-radioactive electrophoretic mobility shift assay, in order to analyze HSF regulation in Neurospora crassa. HSF binds to HSE under normal, non-stress conditions and is thus constitutively trimerized. Upon heat shock, the HSF-HSE complex shows a retarded mobility. This was also observed in Saccharomyces cerevisiae, where this mobility shift was shown to be due to HSF phosphorylation [Sorger and Pelham (1988) Cell 54, 855-864]. In N. crassa, HSE-dependent electrophoretic mobility shift is temperature- and time-dependent. Under normal growth conditions, the HSF is located in the cytoplasm as well as in the nucleus. In germinating conidia the HSF shows a retarded mobility typical for heat shock even at normal growth temperatures. No HSF-dependent mobility shift was detectable in aerial hyphae.