Alice Accorsi

The freshwater golden apple snail (Pomacea canaliculata) is emerging as a powerful model organism for regenerative biology, particularly for studying the molecular mechanisms of sensory organ regeneration. They can fully regenerate their camera-type eyes, which have vertebrate-like anatomical features such as retina, cornea, and lens organized in a closed chamber. Coupled with a relatively small diploid genome, genetic tractability, and ease of laboratory maintenance, P. canaliculata offers unique advantages for studies beyond developmental and regenerative biology. This paper presents a comprehensive workflow for generating and maintaining apple snail mutant lines, making these animals one of the very few genetically tractable mollusks available. The possibility of knocking out genes of interest opens the door to understanding the genetic and molecular basis of sensory organ development and regeneration, as well as other aspects of molluscan biology. Here, we provide detailed protocols for the effective maintenance of a large apple snail colony, collection and microinjection of their zygotes, the use of perivitelline fluid for successful ex ovo culture, and techniques for genotyping and maintaining stable mutant lines. By enabling targeted genetic manipulation in this novel model and disseminating methods for functional genomics in mollusks, we aim to encourage the community to grow and adopt apple snails for further studies across biological fields.

The freshwater golden apple snail (Pomacea canaliculata) is emerging as a powerful model organism for regenerative biology, particularly for studying the molecular mechanisms of sensory organ regeneration. They can fully regenerate their camera-type eyes, which have vertebrate-like anatomical features such as retina, cornea, and lens organized in a closed chamber. Coupled with a relatively small diploid genome, genetic tractability, and ease of laboratory maintenance, P. canaliculata offers unique advantages for studies beyond developmental and regenerative biology. This paper presents a comprehensive workflow for generating and maintaining apple snail mutant lines, making these animals one of the very few genetically tractable mollusks available. The possibility of knocking out genes of interest opens the door to understanding the genetic and molecular basis of sensory organ development and regeneration, as well as other aspects of molluscan biology. Here, we provide detailed protocols for the effective maintenance of a large apple snail colony, collection and microinjection of their zygotes, the use of perivitelline fluid for successful ex ovo culture, and techniques for genotyping and maintaining stable mutant lines. By enabling targeted genetic manipulation in this novel model and disseminating methods for functional genomics in mollusks, we aim to encourage the community to grow and adopt apple snails for further studies across biological fields.

Camera-type eyes are complex sensory organs susceptible to irreversible damage. Their repair is difficult to study due to the paucity of camera-type eye regeneration models. Identifying a genetically tractable organism with the ability to fully regenerate complete camera-type eyes would help overcome this difficulty. Here, we introduce the apple snail Pomacea canaliculata, capable of full regeneration of camera-type eyes even after complete resection. We defined anatomical components of P. canaliculata eyes and genes expressed during crucial steps of their regeneration. By exploiting the unique features of this organism, we successfully established stable mutant lines in apple snails. Our studies reveal that, akin to humans, pax6 is indispensable for eye development in apple snails, establishing this as a research organism to unravel the mechanisms of camera-type eye regeneration. This work expands our understanding of complex sensory organ regeneration and offers a way to explore this process.

Regeneration is the remarkable phenomenon through which an organism can regrow lost or damaged parts with fully functional replacements, including complex anatomical structures, such as limbs. In 2019, Development launched its ‘Model systems for regeneration’ collection, a series of articles introducing some of the most popular model organisms for studying regeneration in vivo. To expand this topic further, this Perspective conveys the voices of five expert biologists from the field of regenerative biology, each of whom showcases some less well-known, but equally extraordinary, species for studying regeneration.

Camera-type eyes are complex sensory organs susceptible to irreversible damage. Their repair is difficult to study due to the paucity of camera-type eye regeneration models. Identifying a genetically tractable organism with the ability to fully regenerate complete camera-type eyes would help overcome this difficulty. Here, we introduce the apple snail Pomacea canaliculata , capable of full regeneration of camera-type eyes even after complete resection. We defined anatomical components of P. canaliculata eyes and genes expressed during crucial steps of their regeneration. By exploiting the unique features of this organism, we successfully established the first stable mutant lines in apple snails. Our studies revealed that, akin to humans, pax6 is indispensable for eye development in apple snails, establishing this as a research organism to unravel the mechanisms of camera-type eye regeneration. This work expands our understanding of complex sensory organ regeneration and offers new ways to explore this process.

Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well-characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering, and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell clustering pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and detect changes between different conditions. Therefore, Image3C expands the use of image-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available.

Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell cluster pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and to detect changes in cellular composition between different conditions. Therefore, Image3C expands the use of imaged-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available. Impact statement: Image3C analyzes cell populations through image-based clustering and neural network training, which allows single-cell analysis in research organisms devoid of species-specific reagents or pre-existing knowledge on cell phenotypes.

The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.

Background: The ability to efficiently visualize and manipulate chromosomes is fundamental to understand the genetic architecture of organisms. This can be challenging for many invertebrates because conventional chromosome preparation protocols deal primarily hypotonic conditions tailored for use with vertebrates and rely extensively on cultured cells which are rarely available for invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate model species is needed. Results: We optimized a chromosome preparation protocol and applied it to several planarian species (Phylum: Platyhelminthes), the freshwater apple snail Pomacea canaliculata (Phylum: Mollusca), and the starlet sea anemone Nematostella vectensis (Phylum: Cnidaria). We showed that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with DI water was sufficient for karyotyping. The karyotypes we obtained allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla, this protocol can be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. Conclusion: The simple and inexpensive chromosome preparation protocol reported here can be readily adapted to new invertebrate research organisms in order to expand and accelerate the discovery of new biology in understudied branches of the tree of life.