Crustacean Parhyale Hawaiensis Research Articles

Active cell divisions generate fourfold orientationally ordered phase in living tissue

Morphogenesis, the process through which genes generate form, establishes tissue-scale order as a template for constructing the complex shapes of the body plan. The extensive growth required to build these ordered substrates is fuelled by cell proliferation, which, naively, should destroy order. Understanding how active morphogenetic mechanisms couple cellular and mechanical processes to generate order—rather than annihilate it—remains an outstanding question in animal development. We show that cell divisions are the primary drivers of tissue flow, leading to a fourfold orientationally ordered phase. Waves of anisotropic cell proliferation propagate across the embryo with precise patterning. Defects introduced into the nascent lattice by cell divisions are moved out of the tissue bulk towards the boundary by subsequent divisions. Specific cell proliferation rates and orientations enable cell divisions to organize rather than fluidize the tissue. We observe this using live imaging and tissue cartography to analyse the dynamics of fourfold tissue ordering in the trunk segmental ectoderm of the crustacean Parhyale hawaiensis beginning 72 h after egg lay. The result is a robust, active mechanism for generating global orientational order in a non-equilibrium system that sets the stage for the subsequent development of shape and form.

Behavioral circatidal rhythms require Bmal1 in Parhyale hawaiensis

Organisms living in the intertidal zone are exposed to a particularly challenging environment. In addition to daily changes in light intensity and seasonal changes in photoperiod and weather patterns, they experience dramatic oscillations in environmental conditions due to the tides. To anticipate tides, and thus optimize their behavior and physiology, animals occupying intertidal ecological niches have acquired circatidal clocks. Although the existence of these clocks has long been known, their underlying molecular components have proven difficult to identify, in large part because of the lack of an intertidal model organism amenable to genetic manipulation. In particular, the relationship between the circatidal and circadian molecular clocks, and the possibility of shared genetic components, has been a long-standing question. Here, we introduce the genetically tractable crustacean Parhyale hawaiensis as a system for the study of circatidal rhythms. First, we show that P.hawaiensis exhibits robust 12.4-h rhythms of locomotion that can be entrained to an artificial tidal regimen and are temperature compensated. Using CRISPR-Cas9 genome editing, we then demonstrate that the core circadian clock gene Bmal1 is required for circatidal rhythms. Our results thus demonstrate that Bmal1 is a molecular link between circatidal and circadian clocks and establish P.hawaiensis as a powerful system to study the molecular mechanisms underlying circatidal rhythms and their entrainment.

Distinct gene expression dynamics in developing and regenerating crustacean limbs

Regenerating animals have the ability to reproduce body parts that were originally made in the embryo and subsequently lost due to injury. Understanding whether regeneration mirrors development is an open question in most regenerative species. Here, we take a transcriptomics approach to examine whether leg regeneration shows similar temporal patterns of gene expression as leg development in the embryo, in the crustacean Parhyale hawaiensis. We find that leg development in the embryo shows stereotypic temporal patterns of gene expression. In contrast, the dynamics of gene expression during leg regeneration show a higher degree of variation related to the physiology of individual animals. A major driver of this variation is the molting cycle. We dissect the transcriptional signals of individual physiology and regeneration to obtain clearer temporal signals marking distinct phases of leg regeneration. Comparing the transcriptional dynamics of development and regeneration we find that, although the two processes use similar sets of genes, the temporal patterns in which these genes are deployed are different and cannot be systematically aligned.

Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis.

Emerging research organisms enable the study of biology that cannot be addressed using classical 'model' organisms. New data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-seq to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis and limb development. In addition, we use short- and long-read RNA-seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.

Knockout of crustacean leg patterning genes suggests that insect wings and body walls evolved from ancient leg segments.

The origin of insect wings has long been debated. Central to this debate is whether wings are a novel structure on the body wall resulting from gene co-option, or evolved from an exite (outgrowth; for example, a gill) on the leg of an ancestral crustacean. Here, we report the phenotypes for the knockout of five leg patterning genes in the crustacean Parhyale hawaiensis and compare these with their previously published phenotypes in Drosophila and other insects. This leads to an alignment of insect and crustacean legs that suggests that two leg segments that were present in the common ancestor of insects and crustaceans were incorporated into the insect body wall, moving the proximal exite of the leg dorsally, up onto the back, to later form insect wings. Our results suggest that insect wings are not novel structures, but instead evolved from existing, ancestral structures.

Analysis of the genetically tractable crustacean Parhyale hawaiensis reveals the organisation of a sensory system for low-resolution vision

BackgroundArthropod eyes have diversified during evolution to serve multiple needs, such as finding mates, hunting prey and navigating in complex surroundings under varying light conditions. This diversity is reflected in the optical apparatus, photoreceptors and neural circuits that underpin vision. Yet our ability to genetically manipulate the visual system to investigate its function is largely limited to a single species, the fruit fly Drosophila melanogaster. Here, we describe the visual system of Parhyale hawaiensis, an amphipod crustacean for which we have established tailored genetic tools.ResultsAdult Parhyale have apposition-type compound eyes made up of ~ 50 ommatidia. Each ommatidium contains four photoreceptor cells with large rhabdomeres (R1–4), expected to be sensitive to the polarisation of light, and one photoreceptor cell with a smaller rhabdomere (R5). The two types of photoreceptors express different opsins, belonging to families with distinct wavelength sensitivities. Using the cis-regulatory regions of opsin genes, we established transgenic reporters expressed in each photoreceptor cell type. Based on these reporters, we show that R1–4 and R5 photoreceptors extend axons to the first optic lobe neuropil, revealing striking differences compared with the photoreceptor projections found in related crustaceans and insects. Investigating visual function, we show that Parhyale have a positive phototactic response and are capable of adapting their eyes to different levels of light intensity.ConclusionsWe propose that the visual system of Parhyale serves low-resolution visual tasks, such as orientation and navigation, based on broad gradients of light intensity and polarisation. Optic lobe structure and photoreceptor projections point to significant divergence from the typical organisation found in other malacostracan crustaceans and insects, which could be associated with a shift to low-resolution vision. Our study provides the foundation for research in the visual system of this genetically tractable species.

The \u201camphi\u201d-brains of amphipods: new insights from the neuroanatomy of Parhyale hawaiensis (Dana, 1853)

BackgroundOver the last years, the amphipod crustacean Parhyale hawaiensis has developed into an attractive marine animal model for evolutionary developmental studies that offers several advantages over existing experimental organisms. It is easy to rear in laboratory conditions with embryos available year-round and amenable to numerous kinds of embryological and functional genetic manipulations. However, beyond these developmental and genetic analyses, research on the architecture of its nervous system is fragmentary. In order to provide a first neuroanatomical atlas of the brain, we investigated P. hawaiensis using immunohistochemical labelings combined with laser-scanning microscopy, X-ray microcomputed tomography, histological sectioning and 3D reconstructions.ResultsAs in most amphipod crustaceans, the brain is dorsally bent out of the body axis with downward oriented lateral hemispheres of the protocerebrum. It comprises almost all prominent neuropils that are part of the suggested ground pattern of malacostracan crustaceans (except the lobula plate and projection neuron tract neuropil). Beyond a general uniformity of these neuropils, the brain of P. hawaiensis is characterized by an elaborated central complex and a modified lamina (first order visual neuropil), which displays a chambered appearance. In the light of a recent analysis on photoreceptor projections in P. hawaiensis, the observed architecture of the lamina corresponds to specialized photoreceptor terminals. Furthermore, in contrast to previous descriptions of amphipod brains, we suggest the presence of a poorly differentiated hemiellipsoid body and an inner chiasm and critically discuss these aspects.ConclusionsDespite a general uniformity of amphipod brains, there is also a certain degree of variability in architecture and size of different neuropils, reflecting various ecologies and life styles of different species. In contrast to other amphipods, the brain of P. hawaiensis does not display any striking modifications or bias towards processing one particular sensory modality. Thus, we conclude that this brain represents a common type of an amphipod brain. Considering various established protocols for analyzing and manipulating P. hawaiensis, this organism is a suitable model to gain deeper understanding of brain anatomy e.g. by using connectome approaches, and this study can serve as first solid basis for following studies.

The amphipod crustacean Parhyale hawaiensis: An emerging comparative model of arthropod development, evolution, and regeneration.

Recent advances in genetic manipulation and genome sequencing have paved the way for a new generation of research organisms. The amphipod crustacean Parhyale hawaiensis is one such system. Parhyale are easy to rear and offer large broods of embryos amenable to injection, dissection, and live imaging. Foundational work has described Parhyale embryonic development, while advancements in genetic manipulation using CRISPR‐Cas9 and other techniques, combined with genome and transcriptome sequencing, have enabled its use in studies of arthropod development, evolution, and regeneration. This study introduces Parhyale development and life history, a catalog of techniques and resources for Parhyale research, and two case studies illustrating its power as a comparative research system.This article is categorized under:Comparative Development and Evolution > Evolutionary NoveltiesAdult Stem Cells, Tissue Renewal, and Regeneration > RegenerationComparative Development and Evolution > Model SystemsComparative Development and Evolution > Body Plan Evolution

Multi-view light-sheet imaging and tracking with the MaMuT software reveals the cell lineage of a direct developing arthropod limb.

During development, coordinated cell behaviors orchestrate tissue and organ morphogenesis. Detailed descriptions of cell lineages and behaviors provide a powerful framework to elucidate the mechanisms of morphogenesis. To study the cellular basis of limb development, we imaged transgenic fluorescently-labeled embryos from the crustacean Parhyale hawaiensis with multi-view light-sheet microscopy at high spatiotemporal resolution over several days of embryogenesis. The cell lineage of outgrowing thoracic limbs was reconstructed at single-cell resolution with new software called Massive Multi-view Tracker (MaMuT). In silico clonal analyses suggested that the early limb primordium becomes subdivided into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb-bud formation is associated with spatial modulation of cell proliferation, while limb elongation is also driven by preferential orientation of cell divisions along the proximal-distal growth axis. Cellular reconstructions were predictive of the expression patterns of limb development genes including the BMP morphogen Decapentaplegic.

The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion.

The amphipod crustacean Parhyale hawaiensis is a blossoming model system for studies of developmental mechanisms and more recently regeneration. We have sequenced the genome allowing annotation of all key signaling pathways, transcription factors, and non-coding RNAs that will enhance ongoing functional studies. Parhyale is a member of the Malacostraca clade, which includes crustacean food crop species. We analysed the immunity related genes of Parhyale as an important comparative system for these species, where immunity related aquaculture problems have increased as farming has intensified. We also find that Parhyale and other species within Multicrustacea contain the enzyme sets necessary to perform lignocellulose digestion ('wood eating'), suggesting this ability may predate the diversification of this lineage. Our data provide an essential resource for further development of Parhyale as an experimental model. The first malacostracan genome will underpin ongoing comparative work in food crop species and research investigating lignocellulose as an energy source.

Live imaging reveals the progenitors and cell dynamics of limb regeneration.

Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis. By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis

Hox genes play crucial roles in establishing regional identity along the anterior–posterior axis in bilaterian animals, and have been implicated in generating morphological diversity throughout evolution. Here we report the identification, expression, and initial genomic characterization of the complete set of Hox genes from the amphipod crustacean Parhyale hawaiensis. Parhyale is an emerging model system that is amenable to experimental manipulations and evolutionary comparisons among the arthropods. Our analyses indicate that the Parhyale genome contains a single copy of each canonical Hox gene with the exception of fushi tarazu, and preliminary mapping suggests that at least some of these genes are clustered together in the genome. With few exceptions, Parhyale Hox genes exhibit both temporal and spatial colinearity, and expression boundaries correlate with morphological differences between segments and their associated appendages. This work represents the most comprehensive analysis of Hox gene expression in a crustacean to date, and provides a foundation for functional studies aimed at elucidating the role of Hox genes in arthropod development and evolution.

Codon and Amino Acid Usage Are Shaped by Selection Across Divergent Model Organisms of the Pancrustacea.

In protein-coding genes, synonymous codon usage and amino acid composition correlate to expression in some eukaryotes, and may result from translational selection. Here, we studied large-scale RNA-seq data from three divergent arthropod models, including cricket (Gryllus bimaculatus), milkweed bug (Oncopeltus fasciatus), and the amphipod crustacean Parhyale hawaiensis, and tested for optimization of codon and amino acid usage relative to expression level. We report strong signals of AT3 optimal codons (those favored in highly expressed genes) in G. bimaculatus and O. fasciatus, whereas weaker signs of GC3 optimal codons were found in P. hawaiensis, suggesting selection on codon usage in all three organisms. Further, in G. bimaculatus and O. fasciatus, high expression was associated with lowered frequency of amino acids with large size/complexity (S/C) scores in favor of those with intermediate S/C values; thus, selection may favor smaller amino acids while retaining those of moderate size for protein stability or conformation. In P. hawaiensis, highly transcribed genes had elevated frequency of amino acids with large and small S/C scores, suggesting a complex dynamic in this crustacean. In all species, the highly transcribed genes appeared to favor short proteins, high optimal codon usage, specific amino acids, and were preferentially involved in cell-cycling and protein synthesis. Together, based on examination of 1,680,067, 1,667,783, and 1,326,896 codon sites in G. bimaculatus, O. fasciatus, and P. hawaiensis, respectively, we conclude that translational selection shapes codon and amino acid usage in these three Pancrustacean arthropods.

A Common Cellular Basis for Muscle Regeneration in Arthropods and Vertebrates

Many animals are able to regenerate amputated or damaged body parts, but it is unclear whether different taxa rely on similar strategies. Planarians and vertebrates use different strategies, based on pluripotent versus committed progenitor cells, respectively, to replace missing tissues. In most animals, however, we lack the experimental tools needed to determine the origin of regenerated tissues. Here, we present a genetically tractable model for limb regeneration, the crustacean Parhyale hawaiensis. We demonstrate that regeneration in Parhyale involves lineage-committed progenitors, as in vertebrates. We discover Pax3/7-expressing muscle satellite cells, previously identified only in chordates, and show that these cells are a source of regenerating muscle in Parhyale. These similarities point to a common cellular basis of regeneration, dating back to the common ancestors of bilaterians.

Transgenesis in non-model organisms: the case of Parhyale.

One of the most striking manifestations of Hox gene activity is the morphological and functional diversity of arthropod body plans, segments, and associated appendages. Among arthropod models, the amphipod crustacean Parhyale hawaiensis satisfies a number of appealing biological and technical requirements to study the Hox control of tissue and organ morphogenesis. Parhyale embryos undergo direct development from fertilized eggs into miniature adults within 10 days and are amenable to all sorts of embryological and functional genetic manipulations. Furthermore, each embryo develops a series of specialized appendages along the anterior-posterior body axis, offering exceptional material to probe the genetic basis of appendage patterning, growth, and differentiation. Here, we describe the methodologies and techniques required for transgenesis-based gain-of-function studies of Hox genes in Parhyale embryos. First, we introduce a protocol for efficient microinjection of early-stage Parhyale embryos. Second, we describe the application of fast and reliable assays to test the activity of the Minos DNA transposon in embryos. Third, we present the use of Minos-based transgenesis vectors to generate stable and transient transgenic Parhyale. Finally, we describe the development and application of a conditional heat-inducible misexpression system to study the role of the Hox gene Ultrabithorax in Parhyale appendage specialization. Beyond providing a useful resource for Parhyalists, this chapter also aims to provide a road map for researchers working on other emerging model organisms. Acknowledging the time and effort that need to be invested in developing transgenic approaches in new species, it is all worth it considering the wide scope of experimentation that opens up once transgenesis is established.

The Maternal Transcriptome of the Crustacean Parhyale hawaiensis Is Inherited Asymmetrically to Invariant Cell Lineages of the Ectoderm and Mesoderm

BackgroundThe embryo of the crustacean Parhyale hawaiensis has a total, unequal and invariant early cleavage pattern. It specifies cell fates earlier than other arthropods, including Drosophila, as individual blastomeres of the 8-cell stage are allocated to the germ layers and the germline. Furthermore, the 8-cell stage is amenable to embryological manipulations. These unique features make Parhyale a suitable system for elucidating germ layer specification in arthropods. Since asymmetric localization of maternally provided RNA is a widespread mechanism to specify early cell fates, we asked whether this is also true for Parhyale. A candidate gene approach did not find RNAs that are asymmetrically distributed at the 8-cell stage. Therefore, we designed a high-density microarray from 9400 recently sequenced ESTs (1) to identify maternally provided RNAs and (2) to find RNAs that are differentially distributed among cells of the 8-cell stage.ResultsMaternal-zygotic transition takes place around the 32-cell stage, i.e. after the specification of germ layers. By comparing a pool of RNAs from early embryos without zygotic transcription to zygotic RNAs of the germband, we found that more than 10% of the targets on the array were enriched in the maternal transcript pool. A screen for asymmetrically distributed RNAs at the 8-cell stage revealed 129 transcripts, from which 50% are predominantly expressed in the early embryonic stages. Finally, we performed knockdown experiments for two of these genes and observed cell-fate-related defects of embryonic development.ConclusionsIn contrast to Drosophila, the four primary germ layer cell lineages in Parhyale are specified during the maternal control phase of the embryo. A key step in this process is the asymmetric distribution of a large number of maternal RNAs to the germ layer progenitor cells.

Identification of a putative germ plasm in the amphipod Parhyale hawaiensis

BackgroundSpecification of the germ line is an essential event during the embryonic development of sexually reproducing animals, as germ line cells are uniquely capable of giving rise to the next generation. Animal germ cells arise through either inheritance of a specialized, maternally supplied cytoplasm called 'germ plasm’ or though inductive signaling by somatic cells. Our understanding of germ cell determination is based largely on a small number of model organisms. To better understand the evolution of germ cell specification, we are investigating this process in the amphipod crustacean Parhyale hawaiensis. Experimental evidence from previous studies demonstrated that Parhyale germ cells are specified through inheritance of a maternally supplied cytoplasmic determinant; however, this determinant has not been identified.ResultsHere we show that the one-cell stage Parhyale embryo has a distinct cytoplasmic region that can be identified by morphology as well as the localization of germ line-associated RNAs. Removal of this cytoplasmic region results in a loss of embryonic germ cells, supporting the hypothesis that it is required for specification of the germ line. Surprisingly, we found that removal of this distinct cytoplasm also results in aberrant somatic cell behaviors, as embryos fail to gastrulate.ConclusionsParhyale hawaiensis embryos have a specialized cytoplasm that is required for specification of the germ line. Our data provide the first functional evidence of a putative germ plasm in a crustacean and provide the basis for comparative functional analysis of germ plasm formation within non-insect arthropods.

ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species

The increased throughput and decreased cost of next-generation sequencing (NGS) have shifted the bottleneck genomic research from sequencing to annotation, analysis and accessibility. This is particularly challenging for research communities working on organisms that lack the basic infrastructure of a sequenced genome, or an efficient way to utilize whatever sequence data may be available. Here we present a new database, the Assembled Searchable Giant Arthropod Read Database (ASGARD). This database is a repository and search engine for transcriptomic data from arthropods that are of high interest to multiple research communities but currently lack sequenced genomes. We demonstrate the functionality and utility of ASGARD using de novo assembled transcriptomes from the milkweed bug Oncopeltus fasciatus, the cricket Gryllus bimaculatus and the amphipod crustacean Parhyale hawaiensis. We have annotated these transcriptomes to assign putative orthology, coding region determination, protein domain identification and Gene Ontology (GO) term annotation to all possible assembly products. ASGARD allows users to search all assemblies by orthology annotation, GO term annotation or Basic Local Alignment Search Tool. User-friendly features of ASGARD include search term auto-completion suggestions based on database content, the ability to download assembly product sequences in FASTA format, direct links to NCBI data for predicted orthologs and graphical representation of the location of protein domains and matches to similar sequences from the NCBI non-redundant database. ASGARD will be a useful repository for transcriptome data from future NGS studies on these and other emerging model arthropods, regardless of sequencing platform, assembly or annotation status. This database thus provides easy, one-stop access to multi-species annotated transcriptome information. We anticipate that this database will be useful for members of multiple research communities, including developmental biology, physiology, evolutionary biology, ecology, comparative genomics and phylogenomics.Database URL: asgard.rc.fas.harvard.edu

How does an animal work?

Summary A newly established interdisciplinary research institute in Lyon, France, aims to bridge the cultural divides between evolution, development, and physiology, in order to study how animal functions come into being, starting from the genomes, and not limited to traditional model systems. Michael Gross reports.

Independent migration of cell populations in the early gastrulation of the amphipod crustacean Parhyale hawaiensis

Cells are the principal component of tissues and can drive morphogenesis through dynamic changes in structure and interaction. During gastrulation, the primary morphogenetic event of early development, cells change shape, exchange neighbors, and migrate long distances to establish cell layers that will form the tissues of the adult animal. Outside of Drosophila, little is known about how changes in cell behavior might drive gastrulation among arthropods. Here, we focus on three cell populations that form two aggregations during early gastrulation in the crustacean Parhyale hawaiensis. Using cytoskeletal markers and lineage tracing we observe bottle cells in anterior and visceral mesoderm precursors as gastrulation commences, and find that both Cytochalasin D, an inhibitor of actin polymerization, and ROCKOUT, an inhibitor of Rho-kinase activity, prevent gastrulation. Furthermore, by ablating specific cells, we show that each of the three populations acts independently during gastrulation, confirming previous hypotheses that cell behavior during Parhyale gastrulation relies on intrinsic signals instead of an inductive mechanism.