Evan Tjeerdema

Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K ^1-10 ). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG2 ¹¹ , the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (∼130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.

Despite the reach of in situ hybridization (ISH) in developmental biology, it is rarely used at scale. The major bottleneck is the throughput of the assay, which relies upon labor-intensive manual steps. The goal of this study was to develop a high-throughput, automated hybridization chain reaction (HCR) pipeline for the sea urchin (Lytechinus pictus). Our method, which we term high-throughput (HT)-HCR, can process 192 gene probe sets on whole-mount embryos within 32 h. The physical properties of sea urchin embryos enabled us to utilize a 96-well plate format, miniaturized reaction volumes, a general-purpose robotic liquid handler and automated confocal microscopy. Using this approach, we produced high quality localization data for 101 target genes across three developmental stages. The results reveal the localization of previously undescribed physiological genes, as well as canonical developmental transcription factors. HT-HCR represents an order of magnitude increase in the throughput of spatial expression profiling studies utilizing the sea urchin. This will enable more-sophisticated perturbation analyses and drug-screening efforts in this emerging animal model.

Despite the reach of in situ hybridization (ISH) in developmental biology, it has rarely been used at scale. The major limitation has been the throughput of the assay, which typically relies upon labor intensive manual steps. The goal of this study was to develop a fully automated hybridization chain reaction (HCR) pipeline capable of large-scale gene expression pattern profiling, with dramatically reduced cost and effort, in the sea urchin Lytechinus pictus . Our resulting pipeline, which we term high throughput (HT)-HCR, can process 192 gene probe sets on whole-mount embryos within 32 hours. The unique qualities of the sea urchin embryo enabled us to automate the entire HCR assay in a 96-well plate format, and utilize highly miniaturized reaction volumes, a general purpose robotic liquid handler, and automated confocal microscopy. From this approach we produced high quality localization data for 101 target genes across three developmental stages of L. pictus . The results reveal the localization of previously undescribed physiological genes, as well as canonical developmental transcription factors. HT-HCR represents a log order increase in the rate at which spatial transcriptomic data can be resolved in the sea urchin. This study paves the way for localization of understudied genes and for sophisticated perturbation analysis. Summary Statement: We developed an automated high-throughput HCR pipeline to rapidly map expression of 101 genes in sea urchin embryos, enabling large-scale discovery of novel developmental gene expression patterns.

Stable transgenesis is a transformative tool in model organism biology. Although the sea urchin is one of the oldest animal models in cell and developmental biology, studies in this animal have largely relied on transient manipulation of wild animals, without a strategy for stable transgenesis. Here, we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus, and establish a robust transgene integration method. Three commonly used transposons (Minos, Tol2 and piggyBac) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression beyond metamorphosis. F0 animals were raised to sexual maturity, and spawned to determine germline integration and transgene inheritance frequency, and to characterize expression patterns of the transgene in F1 progeny. The results demonstrate transgene transmission through the germline, the first example of a germline transgenic sea urchin and, indeed, of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility and efficiency of sea urchin research.

Stable transgenesis is a transformative tool in model organism biology. While the sea urchin is one of the oldest animal models in cell and developmental biology, it has relied on transient manipulations of wild animals, and has lacked a strategy for stable transgenesis. Here we build on recent progress to develop a more genetically tractable sea urchin species, Lytechinus pictus , to establish a robust transgene integration method. Three commonly used transposons ( Minos, Tol2, piggyBac ) were tested for non-autonomous transposition, using plasmids containing a polyubiquitin promoter upstream of a H2B-mCerulean nuclear marker. Minos was the only transposable element that resulted in significant expression past metamorphosis. F ₀ animals were raised to sexual maturity and spawned to determine germline integration, transgene inheritance frequency, and to characterize expression patterns of the transgene in F ₁ progeny. The results demonstrated transgene transmission through the germline, the first example of a germline transgenic sea urchin, and indeed of any echinoderm. This milestone paves the way for the generation of diverse transgenic resources that will dramatically enhance the utility, reproducibility, and efficiency of sea urchin research. Significance Statement: Transgenic tools are essential for effective utilization of animal models. Despite being an established model for cell and developmental biology, the sea urchin has not previously benefited from transgenic technology. This study reports the generation of the first germline transgenic sea urchin and opens new avenues for this organism.

A defining feature of sea urchins is their extreme fecundity. Urchins produce millions of transparent, synchronously developing embryos, ideal for spatial and temporal analysis of development. This biological feature has been effectively utilized for ensemble measurement of biochemical changes. However, it has been underutilized in imaging studies, where single embryo measurements are used. Here we present an example of how stable genetics and high content imaging, along with machine learning‐based image analysis, can be used to exploit the fecundity and synchrony of sea urchins in imaging‐based drug screens. Building upon our recently created sea urchin ABCB1 knockout line, we developed a high‐throughput assay to probe the role of this drug transporter in embryos. We used high content imaging to compare accumulation and toxicity of canonical substrates and inhibitors of the transporter, including fluorescent molecules and antimitotic cancer drugs, in homozygous knockout and wildtype embryos. To measure responses from the resulting image data, we used a nested convolutional neural network, which rapidly classified embryos according to fluorescence or cell division. This approach identified sea urchin embryos with 99.8% accuracy and determined two‐cell and aberrant embryos with 96.3% and 89.1% accuracy, respectively. The results revealed that ABCB1 knockout embryos accumulated the transporter substrate calcein 3.09 times faster than wildtypes. Similarly, knockouts were 4.71 and 3.07 times more sensitive to the mitotic poisons vinblastine and taxol. This study paves the way for large scale pharmacological screens in the sea urchin embryo.