Ancient Circuits, New Resolution: A Single-Cell Atlas of Olfactory Neuron Types Spanning 550 Million Years of Evolution

RESEARCHHUB PREREGISTRATION PROPOSAL

PI: Jorge L. Perez-Moreno, Ph.D.

Katz Lab, Department of Biology, University of Massachusetts Amherst

1. Overview

Scientific rationale: Molluscan nervous systems have yielded some of the most consequential discoveries in the history of neuroscience. Eric Kandel received the Nobel Prize for his work on the molecular basis of memory and long-term potentiation in the sea slug Aplysia californica¹, while Alan Hodgkin and Andrew Huxley received the Nobel Prize for elucidating the ionic mechanism of action potentials using the giant axon of the squid Loligo². These landmark discoveries remain foundational precisely because molluscan neurons reveal conserved principles of neural function in experimentally accessible preparations. Today, single-cell genomics presents an opportunity to extend this tradition into sensory neuroscience: by resolving the molecular identities of individual neuron types and their supporting cells in molluscan chemosensory organs, we can identify the ancestral neural cell programs that underpin olfaction across all animals, including humans. This proposal will generate the first single-cell transcriptomic atlas of a gastropod chemosensory organ using the nudibranch Berghia stephanieae, and computationally identify conserved olfactory neuron-type gene modules shared with mammals. Critically, the PI’s current laboratory has already generated single-cell RNA sequencing data from the Berghia brain, including the rhinophore ganglia and a rhinophore ganglion connectome, enabling direct integration of peripheral sensory neuron atlases with their central neural targets.

Funding and facilities: All work will be conducted at UMass Amherst using the Katz Lab’s established Berghia culture colony, existing dissection and tissue preparation infrastructure, and institutional access to a 10x Genomics Chromium controller and Illumina sequencing through the UMass Genomics Resource Laboratory. Tissue dissociation will be performed using a Cytiva VIA Extractor tissue disaggregator, which the PI has already tested successfully on Berghia tissues. Computational analysis will use the PI’s existing bioinformatics pipelines, including the MolluscaGenes database and custom annotation tools (github.com/invertome). No animal ethics approval is required for invertebrate research under U.S. federal regulations, though all work follows institutional best practices for invertebrate welfare.

Timeline: 12 months from funding to manuscript submission (see Methods for detailed timeline). This includes a 2-month buffer for sequencing turnaround and iterative analysis.

Data sharing commitment: All raw sequencing data will be deposited to NCBI GEO/SRA upon manuscript submission. Processed cell-type atlases, marker gene lists, and cross-species conservation scores will be released as interactive datasets on the MolluscaGenes web platform and archived on Zenodo under CC-BY 4.0 license. Analysis code will be deposited on GitHub (github.com/invertome) with full documentation for reproducibility. All results, including null findings, will be reported as a preprint on bioRxiv concurrent with journal submission, and the manuscript will be submitted to an open access venue.

2. Introduction

Theoretical Background

The sense of smell is arguably the most ancient and universal sensory modality, yet we lack a complete understanding of the fundamental molecular and cellular logic that builds an olfactory system. In vertebrates, the olfactory epithelium contains multiple neuron subtypes, sustentacular support cells, basal progenitor cells, and immune infiltrates, creating a cellular complexity that makes it difficult to disentangle conserved design principles from mammalian-specific elaborations^3,4. In parallel, insect olfactory systems (the antennal lobe) have been mapped at single-cell resolution, revealing organizational principles such as chemotopic receptor-to-glomerulus mapping⁵. Yet whether these principles reflect independent convergence or deep homology remains unresolved, largely because the third great bilaterian clade, Spiralia/Lophotrochozoa, is almost entirely uncharacterized at the olfactory cell-type level.

Molluscs occupy a pivotal phylogenetic position for addressing this gap. As members of Lophotrochozoa, they are equidistant from both vertebrates (Deuterostomia) and insects (Ecdysozoa), making them the critical outgroup for polarizing whether shared olfactory features reflect deep bilaterian homology or independent convergence. Moreover, decades of research on molluscan models such as Aplysia and Loligo have consistently demonstrated that molluscan neurons use the same conserved molecular machinery as mammalian neurons^1,2. We propose that this principle extends to olfactory cell types: that the molecular programs defining chemosensory neurons, support cells, and progenitors in gastropods will share deep homology with their mammalian counterparts, providing a simplified system in which to extract conserved olfactory design principles that are obscured in complex vertebrate tissues.

The nudibranch Berghia stephanieae is an ideal model for this approach. Berghia possesses paired rhinophores, which are chemosensory appendages with glomerular-like neuropil organization strikingly convergent with vertebrate olfactory bulbs and insect antennal lobes⁶. Each rhinophore contains a rhinophore ganglion that processes primary chemosensory input before relaying it to the cerebral ganglia, recapitulating the peripheral-to-central olfactory circuit organization seen in vertebrates (olfactory epithelium → olfactory bulb → cortex) and insects (antenna → antennal lobe → mushroom body). Our preliminary transcriptomic work has revealed novel gastropod-specific G protein variants and expanded families of GPCRs, ionotropic receptors (IRs), and putative chemotactile receptors (including modified nicotinic acetylcholine receptor [nAChR] subunits) in these organs⁷. Some of these signaling components belong to deeply conserved protein families with orthologs in the human olfactory epithelium, while others appear to be lineage-specific innovations, making Berghia uniquely informative for distinguishing ancestral from derived olfactory cell programs. Crucially, Berghia is laboratory-culturable with a ~60-day generation time, highly fecund, and already supported by an annotated reference genome, the MolluscaGenes database⁸, and—uniquely among lophotrochozoan chemosensory models—existing single-cell transcriptomic and connectomic data from the brain including rhinophore ganglia, providing a direct bridge between peripheral sensory neuron identities and their central processing targets. Additionally, nudibranchs display remarkable regenerative abilities, including regeneration of rhinophores, cerata, and even whole brain ganglia after autotomy or injury⁹, raising the possibility that chemosensory organs undergo continuous neuronal turnover supported by resident progenitor populations. Understanding these regenerative mechanisms at single-cell resolution could illuminate fundamental principles of sensory neuron renewal relevant to mammalian olfactory epithelium repair.

Research Questions and Hypotheses

This study addresses two core questions:

Q1: What are the molecular neuron types and supporting cell populations that compose a gastropod chemosensory organ, and how does this neural cell-type architecture compare to that of mammalian olfactory epithelia?

Q2: Are there conserved neuron-type-specific gene modules, shared between molluscs and mammals, that define the fundamental molecular requirements for building an olfactory neural circuit?

We test the following preregistered hypotheses:

H1: Berghia rhinophores contain molecularly distinct olfactory sensory neuron (OSN) subtypes defined by differential expression of chemoreceptor families (IRs, GPCRs, chemotactile receptors including modified nAChR subunits) and signal transduction components (novel G proteins, second messenger cascades), analogous to the receptor-defined OSN subtypes in vertebrate olfactory epithelium.

H2: A sustentacular/support cell population expressing conserved glial/epithelial gene modules (e.g., glutamate transporters, xenobiotic metabolism enzymes, tight junction proteins) will be identifiable in Berghia rhinophores, homologous to the sustentacular cells of mammalian olfactory epithelium. Identifying conserved support cell identity across such phylogenetic distance would provide fundamental insight into whether olfactory tissue architecture requires dedicated non-neuronal cell types, a question with implications for understanding olfactory epithelium maintenance and repair.

H3: Cross-species integration will reveal a core set of conserved olfactory neuron-type gene modules (≥50 genes per module) shared between Berghia and mammalian olfactory tissues, representing ancestral bilaterian cell programs maintained across >550 million years of independent evolution.

H4 (exploratory): Progenitor or stem-like cell populations will be detectable in Berghia rhinophores, expressing regeneration-associated transcription factors (e.g., Sox, Pax, Notch pathway components) orthologous to those involved in mammalian olfactory epithelial regeneration. Given the extraordinary regenerative capacity of nudibranchs, which can regrow entire rhinophores and cerata, these progenitor populations may be more abundant and transcriptionally active than their mammalian counterparts. Importantly, the Katz laboratory has established protocols for EdU pulse-chase proliferation assays and lineage tracing in Berghia, providing a direct route to functionally validate candidate progenitor populations identified by scRNA-seq. If progenitor clusters are identified, follow-up experiments will use EdU incorporation to confirm active proliferation, HCR in situ hybridization to spatially localize progenitor markers within the rhinophore, and rhinophore amputation/regeneration time-course experiments to track progenitor activation and differentiation during sensory organ regrowth.

Impact Statement

Regardless of whether our hypotheses are confirmed or refuted, this study will produce lasting scientific value. If confirmed, the results will establish that the molecular neuron-type architecture of olfaction is far more deeply conserved than currently appreciated, opening gastropod models for mechanistic dissection of olfactory neural circuit signaling with direct translational relevance to human anosmia and neurodegeneration. Combined with our existing brain scRNA-seq and rhinophore ganglion connectome, confirmed homology would enable the first molecular-to-synaptic olfactory circuit reconstruction in any lophotrochozoan, effectively placing Berghia alongside Drosophila and C. elegans as one of the few animals where olfactory circuit architecture can be understood from molecule to synapse. If refuted, for example if Berghia olfactory neuron types share minimal transcriptomic overlap with mammalian counterparts, this would constitute equally important evidence that convergent olfactory neural circuit architectures (glomerular organization) arose through fundamentally different neuron-type solutions, a finding that would reshape our understanding of neural circuit evolution. In either case, the cell-type atlas itself will be the first for any gastropod sensory organ, filling a major gap in comparative neurobiology and providing a foundational resource for the growing community of researchers working with lophotrochozoan models. All data, code, and analyses will be openly shared to maximize reproducibility and reuse.

3. Methods

Participants/Sample

Target organism and sample: Berghia stephanieae (Gastropoda: Nudibranchia), maintained in the Katz lab’s culture at UMass Amherst. Adults (>30 days post-metamorphosis, sexually mature, 15–25 mm body length) will be used for all experiments.

Inclusion criteria: Healthy, actively feeding adults with intact rhinophores; no signs of tissue damage or infection; fed ad libitum on Exaiptasia diaphana anemones for ≥48 hours prior to dissection to standardize nutritional and metabolic state.

Exclusion criteria: Animals showing autotomy, tissue necrosis, abnormal mucus production, or recent spawning (<24 hours), as these conditions alter transcriptomic profiles.

Sample size justification: We will pool rhinophores and oral tentacles from 20–30 individuals per 10x Chromium run to obtain sufficient cell numbers (target: 5,000–10,000 cells per run after quality filtering). This pooling strategy follows established protocols for small invertebrate single-cell studies^10,11 and is necessitated by the small size of individual rhinophores (~0.5–1 mm). Pooling introduces inter-individual genetic heterogeneity; to mitigate this, we will apply strict inclusion/exclusion criteria to minimize physiological variation and use Scrublet for doublet detection calibrated to multi-individual pools. If sufficient single-nucleotide polymorphism diversity exists among pooled individuals, we will apply genetic demultiplexing (e.g., Vireo/souporcell) to assign cells to individuals and assess inter-individual variability. Mucus-associated ambient RNA, a known concern for opisthobranch tissues, will be specifically evaluated by monitoring mucin gene contamination profiles across clusters and addressed computationally using SoupX or CellBender. We will perform 2 independent 10x Chromium runs (biological replicates from separate animal cohorts), each targeting 3,000–5,000 cells after quality filtering, for a combined target of 6,000–10,000 cells. Two biological replicates are essential for distinguishing genuine cell-type markers from cohort-specific or technical artifacts. Power analysis for scRNA-seq cluster detection¹² indicates that 3,000–5,000 cells are sufficient to detect cell types comprising ≥1% of the population with >95% probability, assuming moderate transcriptomic heterogeneity. This initial atlas will serve as pilot data for subsequent grant applications to expand biological replication and explore additional experimental conditions (e.g., regenerating vs. intact rhinophores).

Materials and Procedures

Tissue dissection: Adults will be anesthetized in isotonic MgCl₂ solution. Rhinophores and oral tentacles will be excised under a dissecting microscope using fine tungsten needles, pooled by tissue type, and immediately processed for dissociation.

Tissue dissociation: Pooled tissues will be dissociated using the Cytiva VIA Extractor tissue disaggregator, a semi-automated system that uses gentle mechanical massaging within a multicompartment pouch combined with enzymatic digestion to produce single-cell suspensions¹³. The VIA Extractor has been shown to produce higher cell viability and lower cellular stress compared to conventional manual dissociation and competing instruments, making it particularly well-suited for delicate invertebrate tissues where cell recovery is limiting. The PI has already validated this instrument on Berghia rhinophore tissue, achieving >80% cell viability with the Omics pouch configuration. Enzymatic cocktails (collagenase/dispase in calcium- and magnesium-free artificial seawater) will be loaded into the VIA Extractor pouches and processed at room temperature (~20°C) for 15–30 minutes. Cell viability will be confirmed by trypan blue exclusion with a target of >85% for 10x loading. Cells will be filtered through a 40 µm strainer and resuspended at the manufacturer-recommended concentration (~1,000 cells/µL).

Single-cell RNA sequencing: Single-cell capture, barcoding, and cDNA library construction will be performed using the 10x Genomics Chromium Next GEM Single Cell 3’ v3.1 kit at the UMass Genomics Resource Laboratory. Libraries will be sequenced on an Illumina NovaSeq 6000 (or equivalent platform) to a target depth of ~50,000 reads per cell, sufficient for gene detection saturation in molluscan transcriptomes based on comparable studies in other marine invertebrates^10,11.

Equipment: Dissecting microscope (Leica M205C), Cytiva VIA Extractor tissue disaggregator with Omics pouch kit, 10x Genomics Chromium Controller (UMass Genomics Core), Illumina NovaSeq 6000 (UMass Genomics Core), standard cell biology consumables.

Planned Analyses

Preprocessing pipeline: Raw FASTQ files will be aligned to the recently released annotated Berghia stephanieae reference genome using Cell Ranger (or STARsolo). The availability of a chromosome-level genome assembly with gene annotations enables standard Cell Ranger workflows, including intronic read counting for enhanced gene detection, and avoids the multi-mapping and isoform collapse issues inherent to transcriptome-only references. Gene models will be supplemented with the PI’s MolluscaGenes annotations⁸ where additional functional annotation or ortholog mapping is needed. Quality filtering: cells with <200 genes, >5,000 genes, or >15% mitochondrial reads will be removed. Ambient RNA correction will be performed using SoupX or CellBender, with particular attention to mucin gene contamination profiles given the mucus-rich nature of nudibranch tissues. Doublet detection will use Scrublet with expected doublet rate scaled to cell loading density.

Clustering and annotation: Filtered count matrices will be normalized (scran or SCTransform), dimensionally reduced (PCA, UMAP), and clustered using the Leiden algorithm in Scanpy. Cluster annotation will proceed by: (1) automated marker gene detection (Wilcoxon rank-sum, Bonferroni-corrected p < 0.05, log2FC > 1); (2) manual curation using known chemosensory markers (IRs, GPCRs, chemotactile receptor candidates, novel G protein variants, olfactory marker protein orthologs, neuropeptide precursors); (3) comparison with published gastropod and molluscan neuron transcriptomes where available; and (4) integration with the lab’s existing Berghia brain scRNA-seq dataset (including rhinophore ganglia) to cross-reference peripheral sensory neuron types with their central targets, enabling identification of putative olfactory projection neuron populations and sensory-to-central neurotransmitter/neuropeptide signatures. Furthermore, the lab’s rhinophore ganglion connectome will provide a synaptic wiring framework onto which molecularly defined neuron types can be mapped, enabling a transcriptome-to-connectome integration analogous to efforts in Drosophila and C. elegans but applied for the first time to a lophotrochozoan olfactory circuit. Annotation tools from the PI’s previous work will be adapted for cell-type-level receptor mapping.

Hypothesis-specific predictions and tests:

H1 test: IF rhinophore scRNA-seq reveals ≥2 transcriptionally distinct neuron clusters differentially expressing chemoreceptor families, THEN H1 is supported. We predict 3–6 OSN subtypes based on the number of IR, GPCR, and chemotactile receptor subfamilies detected in bulk rhinophore transcriptomes. IF all neurons cluster together despite expressing different receptors, THEN H1 is refuted, suggesting a single-neuron-type model with stochastic receptor expression.

H2 test: IF a non-neuronal cluster expresses conserved sustentacular markers (glutamate-aspartate transporter, cytochrome P450 xenobiotic enzymes, claudins/occludins) with clear homologs in human olfactory sustentacular cell signatures¹⁴, THEN H2 is supported. IF support cells are absent or molecularly divergent, this informs our understanding of whether sustentacular cell identity is a vertebrate innovation.

H3 test: Cross-species integration using SAMap¹⁵ (which accounts for gene orthology and expression patterns jointly) will be performed between our Berghia atlas and published mouse olfactory epithelium¹⁶ and human olfactory mucosa^4,14 scRNA-seq datasets. Ortholog assignments will leverage the taxonomically comprehensive MolluscaGenes database⁸, using phylogenetically informed methods (reciprocal best-hit BLAST with synteny validation, gene tree reconciliation, etc.) to ensure accurate cross-phylum gene mapping—a critical step given the >550 million year divergence. As a negative control, we will run the same SAMap integration against a non-olfactory mammalian tissue (e.g., liver or skeletal muscle scRNA-seq) to calibrate alignment score thresholds and establish the expected background similarity for unrelated cell types. IF SAMap alignment scores for olfactory cell-type comparisons significantly exceed negative control scores (alignment score >0.1, permutation p < 0.01), THEN H3 is supported. We will extract shared gene modules using non-negative matrix factorization (NMF) and report module sizes, conservation scores, and GO enrichment. IF alignment scores are non-significant relative to negative controls, this constitutes evidence for convergent rather than homologous cell-type architecture.

H4 test (exploratory): IF a cluster with low differentiation scores (CytoTRACE) and expression of stem cell/progenitor-associated transcription factors (Sox2/Sox9 orthologs, Pax6, Notch/Delta) is identified, we will compare its gene signature with published basal cell and progenitor populations from mammalian olfactory epithelium. Candidate progenitor markers will be validated through EdU pulse-chase experiments (protocols already established in the Katz laboratory) to confirm active proliferation, and HCR in situ hybridization on rhinophore cryosections to confirm spatial localization. If promising progenitor populations are identified, this atlas will provide the foundation for a dedicated follow-up study using rhinophore amputation/regeneration time-course scRNA-seq to characterize progenitor activation dynamics.

Contingent analysis decisions: IF cell viability is insufficient for standard 10x capture (<70% viability despite VIA Extractor optimization), THEN we will switch to single-nucleus RNA sequencing (snRNA-seq) using the 10x Chromium Fixed RNA Profiling kit, which tolerates lower viability. IF total cell recovery is <3,000 cells across both runs, THEN we will supplement clustering with reference-based label transfer from the closest available invertebrate scRNA-seq atlas (e.g., Octopus¹¹ or planarian¹⁷ datasets) rather than relying solely on unsupervised clustering.

Ethics and Data Management

Ethical approval: No IACUC approval is required for research on invertebrates (excluding cephalopods) under U.S. federal regulations. All animals will be handled following UMass institutional guidelines for invertebrate welfare, including appropriate anesthetization before dissection.

Confidentiality: No human subjects data are involved. All biological samples derive from a laboratory culture colony.

Data sharing and archiving: Raw sequencing data (FASTQ) and processed count matrices will be deposited to NCBI GEO/SRA under open access upon manuscript submission. Annotated cell-type atlas objects (AnnData/h5ad format) will be deposited to Zenodo (CC-BY 4.0). Cross-species integration results, marker gene lists, and conservation scores will be available through the MolluscaGenes web portal. All analysis code (Scanpy/Python notebooks, SAMap integration scripts) will be released on GitHub (github.com/invertome) with a permissive open-source license (MIT). A preprint will be posted on bioRxiv concurrent with journal submission. We are committed to the principles of open, reproducible science: all computational workflows will be documented with version-pinned dependencies and containerized environments to ensure full reproducibility.

4. Pilot Data

While no single-cell data have yet been generated for this specific project, the following pilot and preliminary work directly demonstrates feasibility:

Bulk transcriptomics of chemosensory organs: The Katz lab has generated and analyzed bulk RNA-seq data from Berghia rhinophores, oral tentacles, and non-sensory tissues. Differential expression analysis identified >150 candidate chemosensory genes enriched in sensory organs, including novel G protein variants, expanded GPCR and ionotropic receptor (IR) families, and putative chemotactile receptors (modified nAChR subunits). This gene set provides a robust prior for scRNA-seq cluster annotation and quality assessment. Receptor annotation and phylogenetic workflows are available at github.com/invertome/berghia-chemogpcrs.

VIA Extractor dissociation validation: The PI has successfully tested the Cytiva VIA Extractor tissue disaggregator on Berghia rhinophore and oral tentacle tissues, achieving viable single-cell suspensions with >85% viability (trypan blue exclusion). The semi-automated, gentle massaging mechanism of the VIA Extractor is particularly advantageous for these small, delicate tissues, producing more consistent and reproducible cell suspensions than manual trituration. This validation eliminates a major technical risk for the proposed single-cell sequencing experiments.

Bioinformatics infrastructure: The PI has developed the MolluscaGenes transcriptomic database and associated annotation pipelines, which provide curated gene models, ortholog assignments, and functional annotations across molluscan taxa⁸. The PI has also developed CrusTome, a large-scale transcriptome database for Crustacea¹⁸, demonstrating a track record of building community genomic resources. Additional bioinformatics tools developed by the PI include HCR-prober, a pipeline for designing HCR v3.0 in situ hybridization probes (github.com/invertome/hcr-prober), and phylogenetically informed annotation workflows (github.com/invertome/PIA2). These and associated tools will be directly applied to scRNA-seq cluster annotation and cross-species gene module identification.

HCR in situ hybridization validation: The PI has ongoing HCR (hybridization chain reaction) experiments targeting candidate chemosensory genes in Berghia rhinophore cryosections, using probes designed with the HCR-prober pipeline. These spatial validation experiments, while independent of the proposed scRNA-seq, will provide complementary spatial confirmation of cell-type-specific expression patterns identified in the atlas.

Brain single-cell transcriptomics and rhinophore ganglion connectome: The Katz lab has generated scRNA-seq data from the Berghia brain, including the rhinophore ganglia—the primary central processing stations for chemosensory input—and has reconstructed a connectome of the rhinophore ganglion. Together, these datasets provide a uniquely powerful resource for the proposed work: molecular neuron types identified in the rhinophore sensory atlas can be cross-referenced with central neuron types in the brain scRNA-seq, and then mapped onto the synaptic wiring diagram of the rhinophore ganglion connectome. This multi-modal integration (transcriptome → spatial expression → synaptic connectivity) parallels the approach that has transformed circuit neuroscience in Drosophila and C. elegans, but has never been applied to a lophotrochozoan olfactory system. The proposed rhinophore scRNA-seq atlas is the critical missing layer that will complete this molecular-to-circuit pipeline.

Cross-species analysis feasibility: The PI has confirmed that published mouse¹⁶ and human^4,14 olfactory scRNA-seq datasets are publicly available and compatible with SAMap integration workflows. Preliminary ortholog mapping between the Berghia transcriptome and human/mouse proteomes yields >8,000 one-to-one orthologs, sufficient for robust cross-species integration¹⁵.

5. Budget

Itemized anticipated costs (adjustable based on pilot data results):

Item	Cost (USD)	Notes
10x Chromium library prep reagents (3’ v3.1 kit)	$2,500	2 captures (biological replicates)
Illumina sequencing (NovaSeq, ~200M reads)	$3,500	UMass Genomics Core institutional rate
Cytiva VIA Extractor consumables	$1,200	Omics pouches, clamps, enzyme cocktails
Consumables (cell strainers, viability staining, QC)	$1,500	Trypan blue, Countess slides, pipette tips, buffers
Animal maintenance and dissecting supplies	$800	Tungsten needles, MgCl₂, ASW reagents, anemone feed
Contingency / snRNA-seq pivot	$500	Reserve for protocol adaptation if needed
TOTAL	$10,000

Computational analysis (clustering, annotation, cross-species integration) will be performed using existing institutional HPC resources and the PI’s established bioinformatics infrastructure at no additional cost. Personnel costs are not requested; the PI will perform all experimental and computational work.

6. References

1. Kandel, E. R. The molecular biology of memory storage: a dialogue between genes and synapses. Science 294, 1030–1038 (2001).

2. Hodgkin, A. L. & Huxley, A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544 (1952).

3. Mombaerts, P. Axonal wiring in the mouse olfactory system. Annu. Rev. Cell Dev. Biol. 22, 713–737 (2006).

4. Durante, M. A. et al. Single-cell analysis of olfactory neurogenesis and differentiation in adult humans. Nat. Neurosci. 23, 323–326 (2020).

5. Vosshall, L. B. & Stocker, R. F. Molecular architecture of smell and taste in Drosophila. Annu. Rev. Neurosci. 30, 505–533 (2007).

6. Wertz, A., Rössler, W., Obermayer, M. & Bickmeyer, U. Functional neuroanatomy of the rhinophore of Aplysia punctata. Front. Zool. 3, 6 (2006).

7. Perez-Moreno, J. L. et al. Transcriptomics reveals novel Gα signaling and CR-like receptors in molluscan chemosensation (2026).

8. Perez-Moreno, J. L. MolluscaGenes: a transcriptomic database for comparative molluscan genomics. In preparation (2026).

9. Miller, J. A. & Byrne, M. Ceratal autotomy and regeneration in the aeolid nudibranch Phidiana crassicornis and the role of predators. Invertebr. Biol. 119, 167–176 (2000).

10. Sebé-Pedrós, A. et al. Cnidarian cell type diversity and regulation revealed by whole-organism single-cell RNA-seq. Cell 173, 1520–1534 (2018).

11. Styfhals, R. et al. Cell type diversity in a developing octopus brain. Nat. Commun. 13, 928 (2022).

12. Svensson, V. et al. Power analysis of single-cell RNA-sequencing experiments. Nat. Methods 14, 381–387 (2017).

13. Cytiva. VIA Extractor tissue disaggregator: gentle tissue disaggregation for optimized cell viability and yield in single-cell omics research. Application note (2023).

14. Zazhytska, M. et al. Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia. Cell 185, 1052–1064 (2022).

15. Tarashansky, A. J. et al. Mapping single-cell atlases throughout Metazoa unravels cell type evolution. eLife 10, e66747 (2021).

16. Hanchate, N. K. et al. Single-cell transcriptomics reveals receptor transformations during olfactory neurogenesis. Science 350, 1251–1255 (2015).

17. Fincher, C. T. et al. Cell type transcriptome atlas for the planarian Schmidtea mediterranea. Science 360, eaaq1736 (2018).

18. Perez-Moreno, J. L. et al. CrusTome: a transcriptome database resource for large-scale analyses across Crustacea. G3 Genes|Genomes|Genetics 13, jkad098 (2023).

Ancient Circuits, New Resolution: A Single-Cell Atlas of Olfactory Neuron Types Spanning 550 Million Years of Evolution

Ancient Circuits, New Resolution: A Single-Cell Atlas of Olfactory Neuron Types Spanning 550 Million Years of Evolution

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