A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

BACKGROUND: How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum have long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. RESULTS: The final assembled and filtered Berghia genome is comparable to other high-quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes) and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. CONCLUSIONS: Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Egg-laying hormone expression in identified neurons across developmental stages and reproductive states of the nudibranch Berghia stephanieae.

Neuropeptides play essential roles in coordinating reproduction. Egg-laying hormone (ELH) is conserved in genetic sequence and behavioral function across molluscs, where neuronal clusters secrete ELH to modulate and induce egg-laying. Here we investigated ELH in the nudibranch mollusc, Berghia stephanieae. ELH preprohormone gene orthologs, which showed clade-specific differences at the C-terminus of the predicted bioactive peptide, were identified in brain transcriptomes across several nudipleuran species, including B. stephanieae. ELH shares deep homology with the corticotropin-releasing hormone gene family, which has roles broadly in stress response. Injection of synthesized B. stephanieae ELH peptide into mature individuals induced egg-laying. ELH gene expression in the brain and body was mapped using in-situ hybridization chain reaction. Across the adult brain, 300-400 neurons expressed ELH. Twenty-one different cell types were identified in adults, three of which were located unilaterally on the right side, which corresponds to the location of the reproductive organs. Ten cell types were present in pre-reproductive juvenile stages. An asymmetric cluster of approximately 100 small neurons appeared in the right pedal ganglion of late-stage juveniles. Additional neurons in the pleural and pedal ganglia expressed ELH only in adults that were actively laying eggs and sub-adults that were on the verge of doing so, implicating their direct role in reproduction. Outside the brain, ELH was expressed on sensory appendages, including in presumptive sensory neurons. Its widespread expression in the nudibranch B. stephanieae suggests that ELH plays a role beyond reproduction in gastropod molluscs.

Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides.

Cephalopods are renowned for changing the color and pattern of their skin for both camouflage and communication. Yet, we do not fully understand how cephalopods control the pigmented chromatophore organs in their skin and change their body pattern. Although these changes primarily rely on eyesight, we found that light causes chromatophores to expand in excised pieces of Octopus bimaculoides skin. We call this behavior light-activated chromatophore expansion (or LACE). To uncover how octopus skin senses light, we used antibodies against r-opsin phototransduction proteins to identify sensory neurons that express r-opsin in the skin. We hypothesized that octopus LACE relies on the same r-opsin phototransduction cascade found in octopus eyes. By creating an action spectrum for the latency to LACE, we found that LACE occurred most quickly in response to blue light. We fit our action spectrum data to a standard opsin curve template and estimated the λmax of LACE to be 480 nm. Consistent with our hypothesis, the maximum sensitivity of the light sensors underlying LACE closely matches the known spectral sensitivity of opsin from octopus eyes. LACE in isolated preparations suggests that octopus skin is intrinsically light sensitive and that this dispersed light sense might contribute to their unique and novel patterning abilities. Finally, our data suggest that a common molecular mechanism for light detection in eyes may have been co-opted for light sensing in octopus skin and then used for LACE.

Cellular-resolution gene expression mapping reveals organization in the head ganglia of the gastropod, Berghia stephanieae.

Gastropod molluscs such as Aplysia, Lymnaea, and Tritonia have been important for determining fundamental rules of motor control, learning, and memory because of their large, individually identifiable neurons. Yet only a small number of gastropod neurons have known molecular markers, limiting the ability to establish brain-wide structure-function relations. Here we combine high-throughput, single-cell RNA sequencing with in situ hybridization chain reaction in the nudibranch Berghia stephanieae to identify and visualize the expression of markers for cell types. Broad neuronal classes were characterized by genes associated with neurotransmitters, like acetylcholine, glutamate, serotonin, and GABA, as well as neuropeptides. These classes were subdivided by other genes including transcriptional regulators and unannotated genes. Marker genes expressed by neurons and glia formed discrete, previously unrecognized regions within and between ganglia. This study provides the foundation for understanding the fundamental cellular organization of gastropod nervous systems.

A chromosome-level genome for the nudibranch gastropod Berghia stephanieae helps parse clade-specific gene expression in novel and conserved phenotypes.

How novel phenotypes originate from conserved genes, processes, and tissues remains a major question in biology. Research that sets out to answer this question often focuses on the conserved genes and processes involved, an approach that explicitly excludes the impact of genetic elements that may be classified as clade-specific, even though many of these genes are known to be important for many novel, or clade-restricted, phenotypes. This is especially true for understudied phyla such as mollusks, where limited genomic and functional biology resources for members of this phylum has long hindered assessments of genetic homology and function. To address this gap, we constructed a chromosome-level genome for the gastropod Berghia stephanieae (Valdés, 2005) to investigate the expression of clade-specific genes across both novel and conserved tissue types in this species. The final assembled and filtered Berghia genome is comparable to other high quality mollusk genomes in terms of size (1.05 Gb) and number of predicted genes (24,960 genes), and is highly contiguous. The proportion of upregulated, clade-specific genes varied across tissues, but with no clear trend between the proportion of clade-specific genes and the novelty of the tissue. However, more complex tissue like the brain had the highest total number of upregulated, clade-specific genes, though the ratio of upregulated clade-specific genes to the total number of upregulated genes was low. Our results, when combined with previous research on the impact of novel genes on phenotypic evolution, highlight the fact that the complexity of the novel tissue or behavior, the type of novelty, and the developmental timing of evolutionary modifications will all influence how novel and conserved genes interact to generate diversity.

Understanding the dermal light sense in the context of integrative photoreceptor cell biology.

While the concept of a dermal light sense has existed for over a century, little progress has been made in our understanding of the mechanisms underlying dispersed photoreception and the evolutionary histories of dispersed photoreceptor cells. These cells historically have been difficult to locate and positively identify, but modern molecular techniques, integrated with existing behavioral, morphological, and physiological data, will make cell identification easier and allow us to address questions of mechanism and evolution. With this in mind, we propose a new classification scheme for all photoreceptor cell types based on two axes, cell distribution (aggregated vs. dispersed) and position within neural networks (first order vs. high order). All photoreceptor cells fall within one of four quadrants created by these axes: aggregated/high order, dispersed/high order, aggregated/first order, or dispersed/first order. This new method of organization will help researchers make objective comparisons between different photoreceptor cell types. Using integrative data from four major phyla (Mollusca, Cnidaria, Echinodermata, and Arthropoda), we also provide evidence for three hypotheses for dispersed photoreceptor cell function and evolution. First, aside from echinoderms, we find that animals often use dispersed photoreceptor cells for tasks that do not require spatial vision. Second, although there are both echinoderm and arthropod exceptions, we find that dispersed photoreceptor cells generally lack morphological specializations that either enhance light gathering or aid in the collection of directional information about light. Third, we find that dispersed photoreceptor cells have evolved a number of times in Metazoa and that most dispersed photoreceptor cells have likely evolved through the co-option of existing phototransduction cascades. Our new classification scheme, combined with modern investigative techniques, will help us address these hypotheses in great detail and generate new hypothesis regarding the function and evolution of dispersed photoreceptor cells.

Accelerated clearing and molecular labeling of biological tissues using magnetohydrodynamic force.

Techniques used to clear biological tissue for fluorescence microscopy are essential to connect anatomical principles at levels ranging from subcellular to the whole animal. Here we report a simple and straightforward approach to efficiently render opaque tissue samples transparent and show that this approach can be modified to rapidly label intact tissue samples with antibodies for large volume fluorescence microscopy. This strategy applies a magnetohydrodynamic (MHD) force to accelerate the removal of lipids from tissue samples at least as large as an intact adult mouse brain. We also show that MHD force can be used to accelerate antibody penetration into tissue samples. This strategy complements a growing array of tools that enable high-resolution 3-dimensional anatomical analyses in intact tissues using fluorescence microscopy. MHD-accelerated clearing is simple, fast, reliable, inexpensive, provides good thermal regulation, and is compatible with existing strategies for high-quality fluorescence microscopy of intact tissues.

Prolific Origination of Eyes in Cnidaria with Co-option of Non-visual Opsins.

Animal eyes vary considerably in morphology and complexity and are thus ideal for understanding the evolution of complex biological traits [1]. While eyes evolved many times in bilaterian animals with elaborate nervous systems, image-forming and simpler eyes also exist in cnidarians, which are ancient non-bilaterians with neural nets and regions with condensed neurons to process information. How often eyes of varying complexity, including image-forming eyes, arose in animals with such simple neural circuitry remains obscure. Here, we produced large-scale phylogenies of Cnidaria and their photosensitive proteins and coupled them with an extensive literature search on eyes and light-sensing behavior to show that cnidarian eyes originated at least eight times, with complex, lensed-eyes having a history separate from other eye types. Compiled data show widespread light-sensing behavior in eyeless cnidarians, and comparative analyses support ancestors without eyes that already sensed light with dispersed photoreceptor cells. The history of expression of photoreceptive opsin proteins supports the inference of distinct eye origins via separate co-option of different non-visual opsin paralogs into eyes. Overall, our results show eyes evolved repeatedly from ancestral photoreceptor cells in non-bilaterian animals with simple nervous systems, co-opting existing precursors, similar to what occurred in Bilateria. Our study underscores the potential for multiple, evolutionarily distinct visual systems even in animals with simple nervous systems.

The Last Common Ancestor of Most Bilaterian Animals Possessed at Least Nine Opsins.

The opsin gene family encodes key proteins animals use to sense light and has expanded dramatically as it originated early in animal evolution. Understanding the origins of opsin diversity can offer clues to how separate lineages of animals have repurposed different opsin paralogs for different light-detecting functions. However, the more we look for opsins outside of eyes and from additional animal phyla, the more opsins we uncover, suggesting we still do not know the true extent of opsin diversity, nor the ancestry of opsin diversity in animals. To estimate the number of opsin paralogs present in both the last common ancestor of the Nephrozoa (bilaterians excluding Xenoacoelomorpha), and the ancestor of Cnidaria + Bilateria, we reconstructed a reconciled opsin phylogeny using sequences from 14 animal phyla, especially the traditionally poorly-sampled echinoderms and molluscs. Our analysis strongly supports a repertoire of at least nine opsin paralogs in the bilaterian ancestor and at least four opsin paralogs in the last common ancestor of Cnidaria + Bilateria. Thus, the kernels of extant opsin diversity arose much earlier in animal history than previously known. Further, opsins likely duplicated and were lost many times, with different lineages of animals maintaining different repertoires of opsin paralogs. This phylogenetic information can inform hypotheses about the functions of different opsin paralogs and can be used to understand how and when opsins were incorporated into complex traits like eyes and extraocular sensors.