3D-printable tools for developmental biology: Improving embryo injection and screening techniques through 3D-printing technology

Abstract

Abbreviations used: 3D printing, three-dimensional printing; dpf, days post-fertilization; RNAi, ribonucleic acid interference; CRISPR, clustered regularly interspaced short palindromic repeats; GFP, green fluorescent protein; p60 or p35, petri dish 60 mm or 35 mm on diameter; MS-222, tricaine mesylate. ABSTRACTDevelopmental biology requires rapid embryo injections and screening. We applied new affordable high-resolution 3D-printing to create five easily modifiable stamp-mold tools that greatly increase injection and screening speed, while simultaneously reducing the harmful aspects of these processes. We designed two stamps that use different approaches to improve the injection efficiency for two different types of embryo, first for embryos from the snail Crepidula fornicata, and second, for those from the spider Parasteatoda tepidariorum. Both drastically improved injection speeds and embryo survival rates, even in novice hands. The other three tools were designed for rapid side-by-side organism orientating and comparison. The first screening tool allows for optimal imaging in Xenopus laevis tadpoles, while the second and third facilitate rapid high-throughput screening of Xenopus tropicalis tadpoles and Danio rerio juveniles, respectively. These designs can act as templates for many injection or screening applications.

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience

“…Targeted disruption of multiple ASD risk genes of diverse cellular functions in human iPSC-derived NPCs altered the proportion of actively dividing cells. Our findings are generally consistent with a series of individual findings across multiple model systems observing brain size changes for individual ASD risk gene perturbations (Bernier et al, 2014;Hoffman et al, 2016;Zhao et al, 2017;Willsey et al, 2020;Durak et al, 2016;Shen et al, 2015;Katayama et al, 2016) and with clinical observations of gross changes in head size in some individuals with ASD, including in those carrying variants in genes studied here, for example, CHD8, DYRK1A, and POGZ (Ji et al, 2015;Evers et al, 2017;van Bon et al, 2016;Yasin et al, 2019;Bernier et al, 2014;O'Roak et al, 2012a;Ye et al, 2015). The direction and magnitude of forebrain size changes likely depend on the strength of the genetic perturbation, the developmental stage at which size is assayed, and the molecular function of the gene.…”

“…The direction and magnitude of forebrain size changes likely depend on the strength of the genetic perturbation, the developmental stage at which size is assayed, and the molecular function of the gene. For example, here we observe chd8 Xenopus mutants have, on average, smaller telencephalons, whereas patients with CHD8 haploinsufficiency exhibit macrocephaly (Ji et al, 2015;Evers et al, 2017;van Bon et al, 2016;Yasin et al, 2019;Bernier et al, 2014;O'Roak et al, 2012a;Ye et al, 2015). It is well-established that affecting NPC biology during neurogenesis can lead to increased or decreased brain size (Shao et al, 2020;Faheem et al, 2015;Lasser et al, 2018).…”

Developing biomedical engineering technologies for reproductive medicine

“…4.4.2 | 3D printing for embryo injection and screening printable tools have been developed in recent years and some have been commercially available. A device with two stamps was designed and prepared for the increased injection and screening of embryos, 84 potentially acting as a feasible platform for clinical applications. Despite the progress, some biocompatible 3D printing machines were proven to cause developmental toxicity in embryo culture, which was evidenced by Danio rerio embryo culture.…”

Optimization of Zebrafish Larvae Sectioning for Mass Spectrometry Imaging

“…Nelson et al investigated 13 distinct embedding media formulations for adult zebrafish, and they found that the 10% gelatin and 5% CMC (carboxymethyl cellulose) mixture is the optimal embedding medium [ 8 ], however, this gelatin/CMC mixture only remains in liquid form at 37 °C or higher, which is not ideal for preserving the small molecule distribution in zebrafish larvae. Marta et al developed 3D-printable tools that make the placement and positioning of the larvae much more reliable and consistent for improving embryo injection and screening [ 9 ], although, sectioning of the fresh frozen larvae was not utilized in their application. Mariana et al embedded zebrafish larvae in 10% gelatin under 50 °C successfully [ 10 ].…”