Amro Hamdoun

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.

Background: Sea urchins have contributed to knowledge of fertilization, embryonic development, and cell physiology for 150 years. Their evolutionary position, as basal deuterostomes, and their long background in developmental biology motivate establishing a genetically enabled sea urchin species. Because of its relatively short generation time of 4–6 months and ease of culture, our lab has focused on the California sea urchin Lytechinus pictus as a multigenerational model and produced knockout and transgenic lines using this species. To ensure that diverse genetic lines can be preserved, methods must be developed to cryopreserve gametes and embryos. We have previously reported methods for cryopreservation of sperm, but robust methods to preserve embryos remain lacking.Results: Here, we describe a relatively simple method to cryopreserve late gastrulae embryos of L. pictus. Importantly, we show that, after thawing and culturing, the embryos progress through larval development, undergo metamorphosis, and yield juvenile adults, indicating the method is robust.Conclusion: The cryopreservation of embryos is an important advance that will facilitate the biobanking, sharing, and long‐term preservation of diverse genetic lines. This method may also eventually prove useful for cryopreservation of embryos of other marine invertebrates.

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.

The following is the default scale for this protocol for each reagent: 10X Terrific Broth without phosphates: 3 L 100X Magnesium sulfate (MgSO4, 125 mM): 1 L 100X Ammonium sulfate ((NH4)2SO4, 2.5 M): 1 L 20X Potassium phosphate (KH2PO4, 960 mM): 1 L 10X Sodium phosphate (Na2HPO4, 500 mM): 1 L 100X D-Glucose (C6H12O6, 1.60 M): 1 L 10X α-Lactose (C12H24O12, 222 mM): 1 L 50% Glycerol (C3H8O3, 6.84 M, 630 g/L): 1 L 1X Auto-induction Terrific Broth (made fresh from stocks per production run): 1 L If you change the scale of the protocol through protocol.io's scaling feature when running the protocol, the scaled values are as follows: 10X Terrific Broth without phosphates: 100X Magnesium sulfate (MgSO4, 125 mM): 100X Ammonium sulfate ((NH4)2SO4, 2.5 M): 20X Potassium phosphate (KH2PO4, 960 mM): 10X Sodium phosphate (Na2HPO4, 500 mM): 100X D-Glucose (C6H12O6, 1.60 M): 10X α-Lactose (C12H24O12, 222 mM): 50% Glycerol (C3H8O3, 6.84 M, 630 g/L): 1X Auto-induction Terrific Broth (made fresh from stocks per production run): The values for all components throughout the protocol should scale accordingly as well. The changes to the suggested container sizes may not make sense, so choose your container appropriately when changing the scale of the protocol. You can see this part of the abstract again by switching to the "Abstract" tab when running the protocol.

Cell-fate commitment in metazoan development relies on precise gene regulatory programs. This study presents a comprehensive single-cell atlas of gene expression (scRNA-seq), nascent transcription (scSLAM-seq), and chromatin accessibility (scATAC-seq) in the purple sea urchin, Strongylocentrotus purpuratus , from early cleavage to pluteus larva stages. Our findings reveal a dynamic regulatory landscape with extensive usage of distal and intronic regulatory elements, which often exhibit cell-type-specific motifs and accessibility profiles that closely track gene expression. We identify a major wave of zygotic genome activation (ZGA) at the 128-cell stage, coinciding with the loss of developmental plasticity, alongside evidence of restricted, lineage-specific gene activation preceding widespread ZGA. Motif analysis highlights distinct regulatory grammars for these early accessible regions. Regulatory element usage largely clusters by germ layer, indicating shared accessibility among related cell types. We delve into the regulatory intricacies of neurons and skeletogenic cells. Sea urchin neurodevelopment proceeds through three distinct lineages, utilizing transcription factors with conserved roles in mammalian neurogenesis. Surprisingly, skeletogenic cells show significant transcriptional and regulatory diversity across their subpopulations, and we identify novel genes associated with calcification. This research offers unprecedented insights into the dynamic regulatory genome of a non-chordate deuterostome, highlighting both conserved principles of gene regulation and unique features that underscore the sea urchin’s importance as a model for understanding developmental and evolutionary genomics in ecologically critical marine species.

Background: Sea urchins have contributed to knowledge of fertilization, embryonic development and cell physiology for 150 years. Their evolutionary position, as basal deuterostomes, and their long background in developmental biology, motivate establishing a genetically-enabled sea urchin species. Because of its relatively short generation time of 4-6 months and ease of culture, our lab has focused on the California sea urchin Lytechinus pictus as a multigenerational model, and produced knockout and transgenic lines using this species. To ensure that diverse genetic lines can be preserved, methods must be developed to cryopreserve gametes and embryos. We have previously reported methods for cryopreservation of sperm, but robust methods to preserve embryos remained lacking. Results: Here, we describe a relatively simple method to cryopreserve late gastrulae embryos of L. pictus . Importantly we show that, after thawing and culturing, the embryos progress through larval development, undergo metamorphosis and yield juvenile adults, indicating the method is robust. Conclusion: The cryopreservation of embryos is an important advance that will facilitate the biobanking, sharing and long-term preservation of diverse genetic lines. This method may also eventually prove useful for cryopreservation of embryos of other marine invertebrates.

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.

Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes, adopting a shape named ‘scutoid’ that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here, we use live imaging of the sea star embryo coupled with deep learning-based segmentation to dissect the relative contributions of cell density, tissue compaction and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing immediately after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

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.