Bimodal optical and optoacoustic multiview microscope in the frequency-domain.

Hybrid fluorescence and optoacoustic microscopy systems have recently emerged as powerful imaging modalities concurrently capturing both radiative and non-radiative molecular relaxations in biological tissues. Nevertheless, such approaches provide limited information as specimens are imaged exclusively from one side, not permitting the acquisition of their full anatomical, structural, or functional features in multiple views of interest. Herein we present a bimodal optical and optoacoustic multiview (BOOM) cost-efficient microscope operating in the frequency-domain for the comprehensive label-free imaging of established and emerging model organisms. Thus, the capabilities of BOOM microscopy have been proven suitable for highly demanding observations in developmental biology and embryology.

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.

Deep learning-assisted frequency-domain photoacoustic microscopy.

Frequency-domain photoacoustic microscopy (FD-PAM) constitutes a powerful cost-efficient imaging method integrating intensity-modulated laser beams for the excitation of single-frequency photoacoustic waves. Nevertheless, FD-PAM provides an extremely small signal-to-noise ratio (SNR), which can be up to two orders of magnitude lower than the conventional time-domain (TD) systems. To overcome this inherent SNR limitation of FD-PAM, we utilize a U-Net neural network aiming at image augmentation without the need for excessive averaging or the application of high optical power. In this context, we improve the accessibility of PAM as the system's cost is dramatically reduced, and we expand its applicability to demanding observations while retaining sufficiently high image quality standards.

Imaging Parhyale hawaiensis embryogenesis with frequency domain photoacoustic microscopy: A novel tool in developmental biology.

We present the application of a low-cost frequency domain photoacoustic (FDPA) microscope for the label-free imaging of live developing embryos of the crustacean model organism Parhyale hawaiensis. By modulating the intensity of a continuous wave laser source at 9.5 MHz, we achieve the excitation of monochromatic PA waves, which are detected to provide amplitude and phase recordings. The data are subsequently processed to generate accurate maximum amplitude projection and surface reconstructions, delineating the morphological features of the embryos with high resolution and contrast. The findings of this study pave the way for the broader adoption of inexpensive PA diagnostic techniques in developmental biology, shedding light on various fundamental processes in established and emerging model organisms.

Cellular basis of limb morphogenesis.

How the size and shape of developing tissues is encoded in the genome has been a longstanding riddle for biologists. Constituent cells integrate several genetic and mechanical signals to decide whether to divide, die, change shape or position. We review here how morphogenetic cell behaviors contribute to leg formation from imaginal disc epithelia in the insect Drosophila melanogaster, as well as to direct embryonic limb outgrowths in the non-insect pancrustacean Parhyale hawaiensis. Considering the deep conservation of developmental programs for limb patterning among arthropods and other bilaterians, moving forward, it will be exciting to see how these genetic similarities reflect at the cellular and tissue mechanics level.

Regionalized tissue fluidization is required for epithelial gap closure during insect gastrulation.

Many animal embryos pull and close an epithelial sheet around the ellipsoidal egg surface during a gastrulation process known as epiboly. The ovoidal geometry dictates that the epithelial sheet first expands and subsequently compacts. Moreover, the spreading epithelium is mechanically stressed and this stress needs to be released. Here we show that during extraembryonic tissue (serosa) epiboly in the insect Tribolium castaneum, the non-proliferative serosa becomes regionalized into a solid-like dorsal region with larger non-rearranging cells, and a more fluid-like ventral region surrounding the leading edge with smaller cells undergoing intercalations. Our results suggest that a heterogeneous actomyosin cable contributes to the fluidization of the leading edge by driving sequential eviction and intercalation of individual cells away from the serosa margin. Since this developmental solution utilized during epiboly resembles the mechanism of wound healing, we propose actomyosin cable-driven local tissue fluidization as a conserved morphogenetic module for closure of epithelial gaps.

Publisher Correction: Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.

In this Letter, the sentence starting: 'For instance, Tribolium and Drosophila inflated are direct targets of the mesoderm…' has been corrected online; see accompanying Amendment.

Attachment of the blastoderm to the vitelline envelope affects gastrulation of insects.

During gastrulation, physical forces reshape the simple embryonic tissue to form the complex body plans of multicellular organisms1. These forces often cause large-scale asymmetric movements of the embryonic tissue2,3. In many embryos, the gastrulating tissue is surrounded by a rigid protective shell4. Although it is well-recognized that gastrulation movements depend on forces that are generated by tissue-intrinsic contractility5,6, it is not known whether interactions between the tissue and the protective shell provide additional forces that affect gastrulation. Here we show that a particular part of the blastoderm tissue of the red flour beetle (Tribolium castaneum) tightly adheres in a temporally coordinated manner to the vitelline envelope that surrounds the embryo. This attachment generates an additional force that counteracts tissue-intrinsic contractile forces to create asymmetric tissue movements. This localized attachment depends on an αPS2 integrin (inflated), and the knockdown of this integrin leads to a gastrulation phenotype that is consistent with complete loss of attachment. Furthermore, analysis of another integrin (the αPS3 integrin, scab) in the fruit fly (Drosophila melanogaster) suggests that gastrulation in this organism also relies on adhesion between the blastoderm and the vitelline envelope. Our findings reveal a conserved mechanism through which the spatiotemporal pattern of tissue adhesion to the vitelline envelope provides controllable, counteracting forces that shape gastrulation movements in insects.

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.

Non-insect crustacean models in developmental genetics including an encomium to Parhyale hawaiensis.

The impressive diversity of body plans, lifestyles and segmental specializations exhibited by crustaceans (barnacles, copepods, shrimps, crabs, lobsters and their kin) provides great material to address longstanding questions in evolutionary developmental biology. Recent advances in forward and reverse genetics and in imaging approaches applied in the amphipod Parhyale hawaiensis and other emerging crustacean model species have made it possible to probe the molecular and cellular basis of crustacean diversity. A number of biological and technical qualities like the slow tempo and holoblastic cleavage mode, the stereotypy of many cellular processes, the functional and morphological diversity of limbs along the body axis, and the availability of various experimental manipulations, have made Parhyale a powerful system to study normal development and regeneration.

Toll Genes Have an Ancestral Role in Axis Elongation.

One of the key morphogenetic processes used during development is the controlled intercalation of cells between their neighbors. This process has been co-opted into a range of developmental events, and it also underlies an event that occurs in each major group of bilaterians: elongation of the embryo along the anterior-posterior axis [1]. In Drosophila, a novel component of this process was recently discovered by Paré et al., who showed that three Toll genes function together to drive cell intercalation during germband extension [2]. This finding raises the question of whether this role of Toll genes is an evolutionary novelty of flies or a general mechanism of embryonic morphogenesis. Here we show that the Toll gene function in axis elongation is, in fact, widely conserved among arthropods. First, we functionally demonstrate that two Toll genes are required for cell intercalation in the beetle Tribolium castaneum. We then show that these genes belong to a previously undescribed Toll subfamily and that members of this subfamily exhibit striped expression (as seen in Tribolium and previously reported in Drosophila [3-5]) in embryos of six other arthropod species spanning the entire phylum. Last, we show that two of these Toll genes are required for normal morphogenesis during anterior-posterior embryo elongation in the spider Parasteatoda tepidariorum, a member of the most basally branching arthropod lineage. From our findings, we hypothesize that Toll genes had a morphogenetic function in embryo elongation in the last common ancestor of all arthropods, which existed over 550 million years ago.

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.

Tribolium embryo morphogenesis: may the force be with you.

Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis.

Cell and tissue dynamics during Tribolium embryogenesis revealed by versatile fluorescence labeling approaches.

Studies on new arthropod models such as the beetle Tribolium castaneum are shifting our knowledge of embryonic patterning and morphogenesis beyond the Drosophila paradigm. In contrast to Drosophila, Tribolium embryos exhibit the short-germ type of development and become enveloped by extensive extra-embryonic membranes, the amnion and serosa. The genetic basis of these processes has been the focus of active research. Here, we complement genetic approaches with live fluorescence imaging of Tribolium embryos to make the link between gene function and morphogenetic cell behaviors during blastoderm formation and differentiation, germband condensation and elongation, and extra-embryonic development. We first show that transient labeling methods result in strong, homogeneous and persistent expression of fluorescent markers in Tribolium embryos, labeling the chromatin, membrane, cytoskeleton or combinations thereof. We then use co-injection of fluorescent markers with dsRNA for live imaging of embryos with disrupted caudal gene function caused by RNA interference. Using these approaches, we describe and compare cell and tissue dynamics in Tribolium embryos with wild-type and altered fate maps. We find that Tribolium germband condensation is effected by cell contraction and intercalation, with the latter being dependent on the anterior-posterior patterning system. We propose that germband condensation drives initiation of amnion folding, whereas expansion of the amniotic fold and closure of the amniotic cavity are likely driven by contraction of an actomyosin cable at the boundary between the amnion and serosa. Our methodology provides a comprehensive framework for testing quantitative models of patterning, growth and morphogenetic mechanisms in Tribolium and other arthropod species.

Identification of DNA sequences that flank a known region by inverse PCR.

The Polymerase Chain Reaction (PCR) with its multiple applications in molecular genetic analysis is the cornerstone of modern basic and applied biomedical research. This chapter focuses on the inverse PCR technique that has been used widely over the last two decades in genotyping and chromosome walking applications for the isolation of unknown DNA sequences upstream and downstream of a known DNA region. The method is based on the use of circularized templates and primers facing outward from the known sequence, rather than primers facing each other used in conventional PCR. As a result, the original genome sequence is rearranged, and stretches of known sequence end up flanking the unknown DNA sequence in the inverse PCR product. I also discuss the special case of using outward facing primers to isolate the intergenic region between genes clustered in tandem or inverted arrangement, since it can hugely simplify the cloning of cis-regulatory sequences in new species of interest.

Reconfiguring gene traps for new tasks using iTRAC.

We recently developed integrase-mediated trap conversion (iTRAC) as a means of exploiting gene traps to create new genetic tools, such as markers for imaging, drivers for gene expression and landing sites for gene and chromosome engineering. The principle of iTRAC is simple: primary gene traps are generated with transposon vectors carrying φC31 integrase docking sites, which are subsequently utilized to integrate different constructs into the selected trapped loci. Thus, iTRAC allows us to reconfigure selected traps for new purposes. Two features make iTRAC an attractive approach for Drosophila research. First, its versatility permits the exploitation of gene traps in an open-ended way, for applications that were not envisaged during the primary trapping screen. Second, iTRAC is readily transferable to new species and provides a means for developing complex genetic tools in drosophilids that lack the facility of Drosophila melanogaster genetics.

A versatile strategy for gene trapping and trap conversion in emerging model organisms.

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis.

Hox genes encode highly conserved transcription factors that regionalize the animal body axis by controlling complex developmental processes. Although they are known to operate in multiple cell types and at different stages, we are still missing the batteries of genes targeted by any one Hox gene over the course of a single developmental process to achieve a particular cell and organ morphology. The transformation of wings into halteres by the Hox gene Ultrabithorax (Ubx) in Drosophila melanogaster presents an excellent model system to study the Hox control of transcriptional networks during successive stages of appendage morphogenesis and cell differentiation. We have used an inducible misexpression system to switch on Ubx in the wing epithelium at successive stages during metamorphosis--in the larva, prepupa, and pupa. We have then used extensive microarray expression profiling and quantitative RT-PCR to identify the primary transcriptional responses to Ubx. We find that Ubx targets range from regulatory genes like transcription factors and signaling components to terminal differentiation genes affecting a broad repertoire of cell behaviors and metabolic reactions. Ubx up- and down-regulates hundreds of downstream genes at each stage, mostly in a subtle manner. Strikingly, our analysis reveals that Ubx target genes are largely distinct at different stages of appendage morphogenesis, suggesting extensive interactions between Hox genes and hormone-controlled regulatory networks to orchestrate complex genetic programs during metamorphosis.

Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in crustacean appendage morphology.

Crustaceans possess remarkably diverse appendages, both between segments of a single individual as well as between species. Previous studies in a wide range of crustaceans have demonstrated a correlation between the anterior expression boundary of the homeotic (Hox) gene Ultrabithorax (Ubx) and the location and number of specialized thoracic feeding appendages, called maxillipeds. Given that Hox genes regulate regional identity in organisms as diverse as mice and flies, these observations in crustaceans led to the hypothesis that Ubx expression regulates the number of maxillipeds and that evolutionary changes in Ubx expression have generated various aspects of crustacean appendage diversity. Specifically, evolutionary changes in the expression boundary of Ubx have resulted in crustacean species with either 0, 1, 2, or 3 pairs of thoracic maxillipeds. Here we test this hypothesis by altering the expression of Ubx in Parhyale hawaiensis, a crustacean that normally possesses a single pair of maxillipeds. By reducing Ubx expression, we can generate Parhyale with additional maxillipeds in a pattern reminiscent of that seen in other crustacean species, and these morphological alterations are maintained as the animals molt and mature. These results provide critical evidence supporting the proposition that changes in Ubx expression have played a role in generating crustacean appendage diversity and lend general insights into the mechanisms of morphological evolution.