Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond

Perillo, Margherita; Paganos, Periklis; Spurrell, Maxwell; Arnone, Maria Ina; Wessel, Gary M.

doi:10.1007/978-1-0716-0974-3_12

Cited by 32 publications

(24 citation statements)

References 23 publications

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“…Fluorescent in situ hybridization was performed as outlined in Perillo et al, 2021 . Fluorescent signal was developed via using fluorophore conjugated tyramide technology (Perkin Elmer, Cat.…”

Section: Methodsmentioning

confidence: 99%

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Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Paganos

Voronov

Musser

et al. 2021

eLife

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Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

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Section: Methodsmentioning

confidence: 99%

“…Immunohistochemical staining or IHC paired with FISH was performed as described in Perillo et al, 2021 . Briefly three dpf plutei were fixed in 4 % paraformaldehyde (PFA) in filtered sea water (FSW) for 15 min at room temperature (RT).…”

Section: Methodsmentioning

confidence: 99%

Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Paganos

Voronov

Musser

et al. 2021

eLife

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“…Immunohistochemical staining or IHC paired with FISH was performed as described in (Perillo et al, 2021). Briefly 3 dpf plutei were fixed in 4 % paraformaldehyde (PFA) in filtered sea water (FSW) for 15 min at room temperature (RT).…”

Section: Methodsmentioning

confidence: 99%

Single cell RNA sequencing of theStrongylocentrotus purpuratuslarva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Paganos

Voronov

Musser³

et al. 2021

Preprint

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Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identified 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these revealed a highly detailed portrait of cell diversity across the larva, including the identification of 12 distinct neuronal cell types. Moreover, we corroborated co-expression of key regulatory genes previously shown to drive sea urchin gene regulatory networks, and revealed additional domains in which these regulatory networks are likely to function within the larva. Lastly, we recovered a neuronal cell type co-expressing Pdx-1 and Brn1/2/4, which had previously been shown to share similar gene expression with vertebrate pancreas. Our results extend this finding, revealing twenty transcription factors shared by this population of neurons in sea urchin and vertebrate pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we generate a draft of the Pdx-1 regulatory network in these cells, and hypothesize this network was present in an ancestral deuterostome neuron before being co-opted into the pancreas developmental lineage in vertebrates.

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“…Fluorescent whole mount in situ hybridization (FISH) was performed as previously described [30]. Embryos were collected and left in fixation solution (4% paraformaldehyde, 32.5% sea water, 32.5 mM MOPS pH 7, 162.5 mM NaCl) overnight at 4°C, then washed in MOPS and stored in 70% ethanol.…”

Section: Star Methodsmentioning

confidence: 99%

Polarized Dishevelled dissolution and condensation drives embryonic axis specification in oocytes

Swartz

Tan

Perillo

et al. 2021

Preprint

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The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species, but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-condensation mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially-regulated Dishevelled dynamics.

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Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond

Cited by 32 publications

References 23 publications

Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Single cell RNA sequencing of theStrongylocentrotus purpuratuslarva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

Polarized Dishevelled dissolution and condensation drives embryonic axis specification in oocytes

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