Visualization of Gli Activity in Craniofacial Tissues of Hedgehog-Pathway Reporter Transgenic Zebrafish

The Hedgehog (Hh)-signaling pathway plays a crucial role in the development and maintenance of multiple vertebrate and invertebrate organ systems. Gli transcription factors are regulated by Hh-signaling and act as downstream effectors of the pathway to activate Hh-target genes. Understanding the requirements for Hh-signaling in organisms can be gained by assessing Gli activity in a spatial and temporal fashion.We have generated a Gli-dependent (Gli-d) transgenic line, Tg(Gli-d:mCherry), that allows for rapid and simple detection of Hh-responding cell populations in both live and fixed zebrafish. This transgenic line expresses a mCherry reporter under the control of a Gli responsive promoter, which can be followed by using fluorescent microscopy and in situ hybridization. Expression of the mCherry transgene reporter during embryogenesis and early larval development faithfully replicated known expression domains of Hh-signaling in zebrafish, and abrogating Hh-signaling in transgenic fish resulted in the suppression of reporter expression. Moreover, ectopic shh expression in Tg(Glid:mCherry) fish led to increased transgene production. Using this transgenic line we investigated the nature of Hh-pathway response during early craniofacial development and determined that the neural crest skeletal precursors do not directly respond to Hh-signaling prior to 48 hours post fertilization, suggesting that earlier requirements for pathway activation in this population of facial skeleton precursors are indirect.We have determined that early Hh-signaling requirements in craniofacial development are indirect. We further demonstrate the Tg(Gli-d:mCherry) fish are a highly useful tool for studying Hh-signaling dependent processes during embryogenesis and larval stages

Action to protect the independence and integrity of global health research

Storeng KT, Abimbola S, Balabanova D, et al. Action to protect the independence and integrity of global health research. BMJ GLOBAL HEALTH. 2019;4(3): e001746

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Optimisation of mesh enclosures for nursery rearing of juvenile sea cucumbers

Mariculture of tropical sea cucumbers is promising, but the nursery rearing of juveniles is a bottleneck for farming and sea ranching. We conducted four medium-scale experiments lasting 3–6 weeks, using thousands of cultured juvenile sandfish Holothuria scabra, to optimise nursery rearing in mesh enclosures in earthen seawater ponds and to test rearing in enclosures in the sea. In one experiment, survival in fine-mesh enclosures (1 m3; 660-µm mesh) related nonlinearly to juvenile size, revealing a threshold body length of 5–8 mm for initial transfer from hatchery tanks. Survival in enclosures within ponds in the other experiments ranged from 78–97%, and differences in growth rates among experiments were explained largely by seasonal differences in seawater temperatures in ponds. Stripped shadecloth units within fine-mesh enclosures increased feeding surfaces and improved growth rates by \u3e15%. On the other hand, shading over the enclosures may lower growth rates. Following the rearing in fine-mesh enclosures, small juveniles (0.5 to 1 g) were grown to stocking size (3–10 g) in coarse-mesh enclosures of 1-mm mesh. Sand or mud added to coarse-mesh enclosures did not significantly improve growth compared to controls without sediment. Survival of sandfish juveniles in coarse-mesh enclosures set on the benthos within seagrass beds differed between two sheltered bays and growth was slow compared to groups within the same type of enclosures in an earthen pond. Our findings should lead to significant improvement in the cost-effectiveness of rearing sandfish juveniles to a stocking size compared to established methods and highlight the need for further research into nursery systems in the sea

The Genetics of Fetal Alcohol Spectrum Disorders

The term Fetal Alcohol Spectrum Disorders (FASD) defines the full range of ethanol-induced birth defects. Numerous variables influence the phenotypic outcomes of embryonic ethanol exposure. Among these variables, genetics appears to play an important role yet our understanding of the genetic predisposition to FASD is still in its infancy