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1. High-throughput genetic manipulation of multicellular organisms using a machine-vision guided embryonic microinjection robot

   https://doi.org/10.1093/genetics/iyae025  

   Alegria, Andrew_D; Joshi, Amey_S; Mendana, Jorge_Blanco; Khosla, Kanav; Smith, Kieran_T; Auch, Benjamin; Donovan, Margaret; Bischof, John; Gohl, Daryl_M; Kodandaramaiah, Suhasa_B; et al (February 2024, GENETICS)

   Abstract Microinjection is a technique used for transgenesis, mutagenesis, cell labeling, cryopreservation, and in vitro fertilization in multiple single and multicellular organisms. Microinjection requires specialized skills and involves rate-limiting and labor-intensive preparatory steps. Here, we constructed a machine-vision guided generalized robot that fully automates the process of microinjection in fruit fly (Drosophila melanogaster) and zebrafish (Danio rerio) embryos. The robot uses machine learning models trained to detect embryos in images of agar plates and identify specific anatomical locations within each embryo in 3D space using dual view microscopes. The robot then serially performs a microinjection in each detected embryo. We constructed and used three such robots to automatically microinject tens of thousands of Drosophila and zebrafish embryos. We systematically optimized robotic microinjection for each species and performed routine transgenesis with proficiency comparable to highly skilled human practitioners while achieving up to 4× increases in microinjection throughput in Drosophila. The robot was utilized to microinject pools of over 20,000 uniquely barcoded plasmids into 1,713 embryos in 2 days to rapidly generate more than 400 unique transgenic Drosophila lines. This experiment enabled a novel measurement of the number of independent germline integration events per successfully injected embryo. Finally, we showed that robotic microinjection of cryoprotective agents in zebrafish embryos significantly improves vitrification rates and survival of cryopreserved embryos post-thaw as compared to manual microinjection. We anticipate that the robot can be used to carry out microinjection for genome-wide manipulation and cryopreservation at scale in a wide range of organisms. 

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2. Reducing Cofilin dosage makes embryos resilient to heat stress

   https://doi.org/10.1101/2025.01.02.631102  

   Biel, Natalie; Rashid, Faizan; Natua, Subhashis; Wang, Ting-Yu; Chou, Tsui-Fen; Nguyen, Thu_Vu Phuc; Golding, Ido; Kalsotra, Auinash; Sokac, Anna Marie (January 2025, bioRxiv)

   Abstract In addition to regulating the actin cytoskeleton, Cofilin also senses and responds to environmental stress. Cofilin can promote cell survival or death depending on context. Yet, many aspects of Cofilin’s role in survival need clarification. Here, we show that exposing earlyDrosophilaembryos to mild heat stress (32°C) induces a Cofilin-mediated Actin Stress Response and upregulation of heat- and ER-stress response genes. However, these responses do not alleviate the negative impacts of heat exposure. Instead, heat stressed embryos show downregulation of hundreds of developmental genes, including determinants of the embryonic body plan, and are less likely to hatch as larvae and adults. Remarkably, reducing Cofilin dosage blunts induction of all stress response pathways, mitigates downregulation of developmental genes, and completely rescues survival. Thus, Cofilin intersects with multiple stress response pathways, and modulates the transcriptomic response to heat stress. Strikingly, Cofilin knockdown emerges as a potent pro-survival manipulation for embryos. 

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3. Rapid joule heating improves vitrification based cryopreservation

   https://doi.org/10.1038/s41467-022-33546-9  

   Zhan, Li; Han, Zonghu; Shao, Qi; Etheridge, Michael L.; Hays, Thomas; Bischof, John C. (December 2022, Nature Communications)

   Abstract Cryopreservation by vitrification has far-reaching implications. However, rewarming techniques that are rapid and scalable (both in throughput and biosystem size) for low concentrations of cryoprotective agent (CPA) for reduced toxicity are lacking, limiting the potential for translation. Here, we introduce a joule heating–based platform technology, whereby biosystems are rapidly rewarmed by contact with an electrical conductor that is fed a voltage pulse. We demonstrate successful cryopreservation of three model biosystems with thicknesses across three orders of magnitude, including adherent cells (~4 µm),Drosophila melanogasterembryos (~50 µm) and rat kidney slices (~1.2 mm) using low CPA concentrations (2–4 M). Using tunable voltage pulse widths from 10 µs to 100 ms, numerical simulation predicts that warming rates from 5 × 10⁴to 6 × 10⁸ °C/min can be achieved. Altogether, our results present a general solution to the cryopreservation of a broad spectrum of cellular, organismal and tissue-based biosystems. 

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4. The combined effects of acidification and acute warming on the embryos of Pacific herring (Clupea pallasii)

   https://doi.org/10.3389/fmars.2023.1307617  

   Singh, Nicole R.; Love, Brooke; Murray, Christopher S.; Sobocinski, Kathryn L.; Cooper, W. James (December 2023, Frontiers in Marine Science)

   Anthropogenic climate change is projected to affect marine ecosystems by challenging the environmental tolerance of individuals. Marine fishes may be particularly vulnerable to emergent climate stressors during early life stages. Here we focus on embryos of Pacific herring(Clupea pallasii), an important forage fish species widely distributed across the North Pacific. Embryos were reared under a range of temperatures (10-16°C) crossed with twopCO₂levels (600 and 2000μatm) to investigate effects on metabolism and survival. We further tested how elevatedpCO₂affects critical thermal tolerance (CT_max) by challenging embryos to short-term temperature fluctuations. Experiments were repeated on embryos collected from winter and spring spawning populations to determine if spawning phenology corresponds with different limits of environmental tolerance in offspring. We found that embryos could withstand acute exposure to 20°C regardless of spawning population or incubation treatment, but that survival was greatly reduced after 2-3 hours at 25°C. We found thatpCO₂had limited effects onCT_max. The survival of embryos reared under chronically warm conditions (12°, 14°, or 16°C) was significantly lower relative to 10°C treatments in both populations. Oxygen consumption rates (MO₂) were also higher at elevated temperatures andpCO₂levels. However, heart contraction measurements made 48 hours afterCT_maxexposure revealed a greater increase in heart rate in embryos reared at 10°C compared to 16°C, suggesting acclimation at higher incubation temperatures. Our results indicate that Pacific herring are generally tolerant ofpCO₂but are vulnerable to acute temperature stress. Importantly, spring-spawning embryos did not clearly exhibit a higher tolerance to heat stress compared to winter offspring. 

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5. Drosophila Late Embryonic through Late Larval Stage Body Wall Dissection: Dissection Tools and Techniques

   https://doi.org/10.1101/pdb.prot108499  

   Ashley, James; Carrillo, Robert A (June 2024, Cold Spring Harbor Protocols)

   One of the challenges of studying synaptic structure and function is accessibility. Some of the earliest readily identifiable and accessible synapses were from the frog and various arthropods. To address questions regarding mechanisms that underlie synaptic development and function, genetically tractable systems were required, and researchers turned to theDrosophila melanogasterembryonic/larval neuromuscular preparation.Drosophilaembryos are transparent and can be labeled with antibodies or probes and imaged in whole-mount preparation for structural analysis. Embryos can also be dissected to visualize the entire body wall musculature as well as finer details including live protein trafficking and protein–protein interactions. Whereas younger dissected embryos can be mounted directly onto charged slides, more mature embryos and larvae develop a cuticle that impedes this adherence, so different techniques must be applied. In this protocol, we detail how to manufacture dissection tools and collect embryos, and discuss the individual steps of dissecting late-stage embryos, early first-instar larvae, and late-stage third-instar larvae. 

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